WO2005011632A1 - Liposome dirige sur une cible, enterique et a absorption commandee possedant une chaine de sucre, ainsi que remede contre le cancer contenant ce liposome et diagnostic mettant en oeuvre ce liposome - Google Patents

Liposome dirige sur une cible, enterique et a absorption commandee possedant une chaine de sucre, ainsi que remede contre le cancer contenant ce liposome et diagnostic mettant en oeuvre ce liposome Download PDF

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Publication number
WO2005011632A1
WO2005011632A1 PCT/JP2004/011291 JP2004011291W WO2005011632A1 WO 2005011632 A1 WO2005011632 A1 WO 2005011632A1 JP 2004011291 W JP2004011291 W JP 2004011291W WO 2005011632 A1 WO2005011632 A1 WO 2005011632A1
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WIPO (PCT)
Prior art keywords
ribosome
sugar chain
drugs
liposome
membrane
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PCT/JP2004/011291
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English (en)
Japanese (ja)
Inventor
Noboru Yamazaki
Hideo Tsurushima
Shuuji Kojima
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National Institute Of Advanced Industrial Science And Technology
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Priority to EP04748262A priority Critical patent/EP1655038A4/fr
Priority to US10/566,566 priority patent/US20070160657A1/en
Priority to JP2005512589A priority patent/JPWO2005011632A1/ja
Publication of WO2005011632A1 publication Critical patent/WO2005011632A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14—Liposomes; Vesicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/57—Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances

Definitions

  • the present invention relates to a therapeutic drug delivery system and a drug delivery system for recognizing target cells and tissues such as cancer and locally delivering drugs and genes to an affected area, which can be applied in the field of medicine and cosmetics, including pharmaceuticals and cosmetics.
  • the present invention relates to a sugar chain-modified ribosome which can be used as a cell / tissue sensing probe for diagnosis and has particularly excellent intestinal absorbability, and a ribosome preparation containing a drug or a gene encapsulated therein.
  • DDS drug delivery system
  • NNI National Nanotechnology Strategy
  • DDS drug delivery system
  • One of these is "Establishment of basic seeds for technologies such as biofunctional materials for extending healthy life expectancy and pinpoint treatment.”
  • the morbidity and mortality of cancer are increasing year by year in an aging society, and the development of target-oriented DDS, a new therapeutic material, is expected.
  • the importance of targeted DDS nanomaterials with no side effects in other diseases is attracting attention, and the market size is expected to exceed 10 trillion yen in the near future. These materials are also expected to be used for diagnosis as well as treatment.
  • target-oriented (Targeting) DDS is the concept of sending a drug “to the required site in the body”, “the required amount”, and “for the required time”.
  • liposomes which are fine-particle carriers, are attracting attention.
  • Passive targeting methods such as changing the type, composition ratio, particle size, and surface charge of ribosome lipids, have been attempted to give these particles a target-directing function, but this method is still unsuccessful. Sufficient and further improvement is required.
  • selectins DC-SIGN, DC-SGNR, collectins, C-type lectins such as mannose-binding proteins, I-type lectins such as Siglec, and mannose as receptors present on the cell membrane surface of each target tissue.
  • C-type lectins such as mannose-binding proteins
  • I-type lectins such as Siglec
  • mannose as receptors present on the cell membrane surface of each target tissue.
  • lectins sucgar chain recognition proteins
  • P-type lectin such as 6_phosphate receptor
  • R-type lectin such as 6_phosphate receptor
  • R-type lectin such as 6_phosphate receptor
  • L-type lectin L-type lectin
  • M-type lectin M-type lectin
  • galectin galectin
  • Ribosomes with ligands attached to the outer membrane surface are selected for target sites such as cancer.
  • Many studies have been conducted as DDS materials for selectively delivering drugs and genes. However, most of them bind to target cells in vitro but are not targeted to the expected target cells or filaments in vivo
  • Leukemia is one of the most advanced cancers in the analysis and treatment of its condition.
  • the main treatment is chemotherapy, but the chemotherapy is very powerful because of the possibility of remission (healing).
  • DDS Drug Delivery System
  • DDS material that can be used by oral administration, which is the easiest and cheapest to administer, is also an important issue.
  • peptidic and proteinaceous drugs are generally water-soluble and have high molecular weight and low permeability through the small intestinal mucosa of the gastrointestinal tract. Therefore, research on ligand-binding ribosomes as a DDS material for delivering these high-molecular-weight drugs and genes from the intestinal tract to the blood is being conducted (Lehr, C. -M. (2000) J. Controlled Release). 65, 19-29).
  • studies on intestinal absorption-regulated ribosomes using sugar chains as these ligands have not yet been reported.
  • the present inventors have found that a sugar chain is already bound to a ribosome membrane via a linker protein, and that the sugar chain is a Lewis X-type trisaccharide, a sialyl Lewis X-type tetrasaccharide, a 3, -sialyl ratatosamine trisaccharide. 6'-Sialyl lactosamine trisaccharide, which is ribosome membrane Z or linker protein, to which tris (hydroxymethyl) aminomethane is arbitrarily bound to be hydrophilic.
  • a patent application has been filed for intestinal absorption-controlling ribosome, which is a ribosome modified by a chain, wherein a sugar chain may be bound to the ribosome via a linker protein. Disclosure of the invention
  • the present invention relates to a ribosome having a sugar chain having specific binding activity to various lectins (sugar chain recognition proteins) present on the cell surface of various tissues
  • An object of the present invention is to provide a liposome capable of distinguishing cells and tissues in a living body and efficiently transporting a drug or a gene. Further, the present invention aims to provide a therapeutic agent for a disease containing the ribosome. Another object of the present invention is to provide a ribosome having high stability.
  • the present inventors have studied the composition of ribosomes, and have obtained ribosomes with high stability.
  • various experiments and examinations were conducted on the types and bond densities of the bran chains to be bonded to the ribosome surface, linker proteins, and compounds for hydrolyzing the ribosomes, and the structure of the sugar chains was analyzed for each tissue.
  • the liposome surface and liposome-protein can be hydrated with a specific hydrophilic compound, and the density of sugar chains that bind to liposonym can be controlled to transfer liposomes to each tissue. It has been found that the amount can be further increased, and thereby, the drug or gene can be efficiently transported to a target cell or tissue.
  • the present invention has been completed. ',
  • the present inventors have further conducted intensive studies on the actual use of the ribosomes thus obtained for the treatment of diseases, and determined various types of tissues and organs depending on the types of sugar chains bonded to the surface. The present inventors have found that the present invention can be applied to such diseases, and have completed the present invention.
  • the present inventors have further reduced the toxicity by embedding the anticancer drug doxorubicin, whose toxicity is a problem, in ribosomes.
  • this alone increases drug safety, it also reduces cytotoxicity against leukemia cells.
  • the present invention relates to the following 1 to 79.
  • the liposome is composed of phosphatidylcholines (molar ratio ⁇ 70%), phosphatidylethanolamines (molar ratio 0-30%), phosphatidic acids, long-chain alkyl phosphates and the like. At least one lipid selected from the group consisting of phosphodicetyl phosphates (0 to 30% molar ratio), gandariosides, glycolipids, phosphatidyl glycerols, and phosphodingomyelins One or more lipids (molar ratio 0-40%), and cholesterols (molar ratio 0-70%) The sugar chain-modified ribosome according to 1.
  • the sugar chain-modified ribosome according to any one of 1 to 4, wherein the ribosome has a particle size of 30 to 500 nm.
  • the linker protein is formed from at least one lipid selected from the group consisting of gandariosides, glycolipids, phosphatidylglycerols, sphingomyelins, and cholesterols formed on the ribosome surface. Naru rough 15. The bran chain-modified ribosome according to any one of 1 to 14, which is bound to a ribosome.
  • the compound is hydrophilized by a hydrophilic compound, and the hydrophilic compound is represented by the general formula (1) X-RI (R20H) n formula (1)
  • X represents a reactive functional group that binds to a ribosomal lipid or a linker protein directly or to a bivalent reagent for crosslinking, and n represents a natural number.
  • the compound is hydrophilized by a hydrophilic compound, and the hydrophilic compound is represented by the general formula (2) H, N-R3- (R40H) n formula (2)
  • R3 represents a linear or branched hydrocarbon chain of C1 to C40
  • R4 is absent or linear or branched of C1 to C40.
  • hydrophilized by a hydrophilic compound wherein the hydrophilic compound has the general formula (3) H 2 N-R5 (OH) n formula (3)
  • Ribosomal membrane Z or linker protein is made hydrophilic by covalently bonding a hydrophilic compound that is a tris (hydroxylanoloxy) aminoalkane to ribosome membrane Z or linker protein.
  • a hydrophilic compound that is a tris (hydroxylanoloxy) aminoalkane to ribosome membrane Z or linker protein.
  • Glycosylated ribosomes include selectins, DC-SIGN, DC-SGNR ⁇ , and C-type lectins including mannose-binding lectin and siglec, which are present on the cell membrane surface of each tissue I Type lectin, 1 to 26 targeting lectin selected from the group consisting of P-type lectin containing mannose-6-phosphate receptor, R-type lectin, L-type lectin, M-type lectin and galectin 3. The sugar chain-modified ribosome according to 1.).
  • the sugar chain is bound to the ribosome membrane, and the sugar chain is alpha-1,2 mannobiose disaccharide, alpha 1,3 mannobiose disaccharide, alpha 1,4 mannobiose disaccharide, anorefa 1 , 6 Mannobiose di-nano chain, anorefer 1,3 anorefer 1,6 mannotriose trisaccharide, oligomannose 3 pentasaccharide, oligomannose 4b hexasaccharide, oligomannose 5 heptasaccharide, oligomannose 688 Sugar chain, oligomannose 7-nine sugar chain, oligomannose octa-chain, oligomannose 9-1 "monosaccharide chain, 3 sialyllatose trisaccharide, 6, -sialyllatose trisaccharide, 3'-sialyllatose tosamine trisamine
  • Drugs include alkylating anticancer drugs, antimetabolites, plant-derived anticancer drugs, anticancer antibiotics, BRM * cytokines, platinum complex anticancer drugs, immunotherapy drugs, hormonal anticancer drugs, monoclonal antibodies, etc.
  • Drugs for oncology drugs for central nervous system, drugs for peripheral nervous system Tonic, blood and body fluid
  • Drugs metabolic drugs, antibiotics, chemotherapeutic drugs, test drugs, anti-inflammatory drugs, ophthalmic drugs, central nervous system drugs, autoimmune drugs, cardiovascular drugs, diabetes, hyperlipidemia, etc.
  • the ribosome preparation according to 4. .:
  • the ribosome preparation according to 34 or 35 which is a preparation for oral administration.
  • the ribosome preparation according to 34 or 35 which is a preparation for parenteral administration.
  • 38. The liposome preparation according to 35, wherein the drug contained in the sugar chain-modified ribosome is doxorubicin.
  • the anticancer agent according to 39 which is an anticancer agent for oral administration.
  • the anticancer agent according to 39 which is an anticancer agent for parenteral administration.
  • Liposome whose ribosome membrane is hydrophilic and whose surface does not have sugar chains attached.
  • Liposomes are composed of phosphatidylcholines (molar ratio 0-70%), phosphatidyl ethanolamines (molar ratio 0-30%), phosphatidic acids, and long-chain alkyl phosphates.
  • One or more lipids selected from the group consisting of salts and dicetyl phosphates (molar ratio: 0 to 30%), gandariosides, glycolipids, phosphatidyl glycerols and sphingomyelins 1 42.
  • the liposome according to 42 comprising at least one kind of lipid (molar ratio: 0 to 40%) and cholesterols (molar ratio: 0 to 70 %).
  • the ribosome according to 42 or 43 further comprising a protein.
  • the liposome according to 45, wherein the low-molecular-weight hydrophilic compound is a compound having at least one 0H group.
  • the hydrophilic compound has the general formula (1)
  • R1 represents a C1 to C40 straight or branched hydrocarbon chain
  • R2 is absent or a C1 to C40 linear or branched hydrocarbon chain.
  • X represents a reactive functional group that binds directly to a ribosomal lipid or linker protein or to a bivalent reagent for crosslinking
  • n represents a natural number, a liposome.
  • the hydrophilic compound has the general formula (2)
  • the liposome according to 45 wherein R3 represents a C1 to C40 linear or branched hydrocarbon chain, and R4 is absent or a C1 to C40 linear or branched hydrocarbon chain. shows the chain,.
  • H 2 n was or directly ribosome lipid or linker one protein represents a reactive functional group bonded to a bivalent reagent for crosslinking, n represents a natural number, liposomes.
  • liposome according to 45 wherein R5 represents a C1 to C40 linear or branched hydrocarbon chain, and N represents a divalent reagent for direct or cross-linking with ribosomal lipid or linker protein.
  • Liposo indicates a reactive functional group to be attached, and n indicates a natural number Home.
  • Ribosomal membrane and no or linker protein are made hydrophilic by covalently binding a hydrophilic compound that is a tris (hydroxyalkyl) aminoalkane to the ribosome membrane Z or linker protein.
  • a hydrophilic compound that is a tris (hydroxyalkyl) aminoalkane to the ribosome membrane Z or linker protein.
  • a liposome membrane is formed by covalently bonding a hydrophilic compound selected from the group consisting of aminoaminomethane, tris (hydroxymethyl) minopropane, tris (hydroxyxethyl) aminopropane, and tris (-hydroxypropyl) aminopropane.
  • a hydrophilic compound selected from the group consisting of aminoaminomethane, tris (hydroxymethyl) minopropane, tris (hydroxyxethyl) aminopropane, and tris (-hydroxypropyl) aminopropane.
  • Drugs include alkylating anticancer drugs, antimetabolites, plant-derived anticancer drugs, anticancer antibiotics, BRM * cytokines, platinum complex anticancer drugs, immunotherapy drugs, hormonal anticancer drugs, monoclonal antibodies, etc.
  • Drugs for oncology drugs for central nervous system, drugs for peripheral nervous system, sensory organs, drugs for respiratory diseases, drugs for cardiovascular systems, drugs for digestive organs, drugs for hormonal systems, urology, reproductive organs, drugs for external use, vitamins, nutrition Tonics, drugs for blood and body fluids, metabolic drugs, antibiotics, chemotherapeutic drugs, test drugs, anti-inflammatory drugs, ophthalmic drugs, central nervous system drugs, autoimmune drugs, cardiovascular drugs, diabetes, Drugs for lifestyle-related diseases such as hyperlipidemia or various drugs for oral, pulmonary, transdermal or transmucosal, corticosteroids, immunosuppressants, antibacterials, antivirals, angiogenesis inhibitors , Cytokines, chemokine, anti-cytokine antibodies, anti-chemokine antibodies, anti-cytokines.
  • Chemokine receptor antibodies nucleic acid preparations related to gene therapy such as siRNA, miRNA, smRNA, antisense 0DN and DNA, neuroprotective factors, antibodies 57.
  • the ribosome preparation according to 57 which is selected from the group consisting of pharmaceuticals.
  • the ribosome preparation according to 57 or 58 which is a preparation for oral administration.
  • 60. The ribosome preparation according to 57 or 58, which is a preparation for parenteral administration.
  • An anticancer agent comprising the ribosome preparation according to 58, wherein the drug is a tumor drug.
  • 62. The liposome preparation according to 61, wherein the drug contained in the sugar chain-modified ribosome is doxorubicin.
  • the anticancer agent according to 61 or 62 which is an anticancer agent for oral administration.
  • the anticancer agent according to 61 or 62 which is an anticancer agent for parenteral administration.
  • a cosmetic composition comprising a ribosome preparation in which a cosmetic is encapsulated in the sugar chain-modified ribosome according to any one of 1 to 33. .
  • the cosmetic composition according to 65 which is a preparation for transdermal administration.
  • a food composition comprising a ribosome preparation in which a food is encapsulated in the sugar chain-modified ribosome according to any one of 1 to 33. ',
  • the food composition according to 68 which is a formulation for oral administration.
  • T 1 The food composition according to 69 or 70, wherein the food is vitamin A or vitamin E.
  • a cosmetic composition comprising a liposome preparation in which a cosmetic is encapsulated in the ribosome according to any one of to 42 to 56.
  • the cosmetic composition according to 72 which is a preparation for transdermal administration.
  • a food composition comprising a liposomal preparation in which a food is encapsulated in the ribosome according to any one of to 52 to 56.
  • the food composition according to 75 which is a formulation for oral administration.
  • FIG. 1 is a schematic diagram showing an example of the structure of a ribosome to which alpha 1,2-mannobiose disaccharide is linked.
  • -FIG. 2 is a schematic diagram showing an example of the structure of a ribosome to which an alpha 1,3 mannobiose disaccharide is linked.
  • FIG. 3 is a schematic diagram showing an example of the structure of a ribosome to which alpha 1,4 mannobiose disaccharide is linked.
  • FIG. 4 is a schematic diagram showing an example of the structure of a liposonim to which an alpha 1,6 mannobiose disaccharide is bound.
  • FIG. 5 is a schematic diagram showing an example of the structure of a liposome bound to alpha 1,3 alpha 1,6 mannotriose trisaccharide. ⁇ ',
  • FIG. 6 is a schematic diagram showing a structural example of a ribosome to which oligomannose 3 pentasaccharide is bound.
  • FIG. 7 is a schematic diagram showing a structural example of a ribosome having oligomannose 4b hexasaccharide linked thereto.
  • FIG. 8 is a schematic diagram showing an example of the structure of a ribosome to which oligomannose 5 heptasaccharides are bound.
  • FIG. 9 is a schematic diagram showing a structural example of a ribosome to which oligomannose 6-octasaccharide is linked.
  • FIG. 10 is a schematic diagram showing a structural example of a ribosome to which oligomannose 7-nine sugar chains are bound.
  • FIG. 11 is a schematic diagram showing a structural example of a ribosome to which oligomannose octasaccharide is bound.
  • FIG. 12 is a schematic diagram showing an example of the structure of a ribosome to which oligomannose 9 H ⁇ -sugar is bound.
  • FIG. 13 is a graph showing the distribution of 13 kinds of liposome complexes in blood 60 minutes after intravenous administration.
  • FIG. 14 is a graph showing the distribution of 13 types of ribosome complexes to the liver 60 minutes after intravenous administration.
  • FIG. 15 is a graph showing the distribution of 13 kinds of liposome complexes to the spleen eo minutes after intravenous administration.
  • FIG. 16 is a diagram showing the distribution of lungs 60 minutes after intravenous administration of 13 kinds of liposome complexes.
  • FIG. 17 is a diagram showing the distribution of 13 ribosome complexes to the brain 60 minutes after intravenous administration.
  • FIG. 18 is a graph showing the distribution of 13 kinds of liposome complexes to cancer tissues 60 minutes after intravenous administration.
  • '-FIG. 19 is a graph showing the distribution of 13 kinds of liposome complexes to lymph nodes 60 minutes after intravenous administration.
  • FIG. 20 is a diagram showing the distribution of thymus distribution 60 minutes after intravenous administration of 13 types of ribosome complexes.
  • FIG. 21 is a diagram showing the distribution of 13 ribosome complexes to the heart 60 minutes after intravenous administration.
  • FIG. 22 is a graph showing the distribution of 13 liposome complexes in the small intestine 60 minutes after intravenous administration.
  • FIG. 23 is a schematic diagram showing an example of the structure of a ribosome to which a 3′-sialyl ratatose trisaccharide is bound.
  • FIG. 24 is a schematic diagram showing an example of the structure of a ribosome to which 6, -carbyllatatose trisaccharide is bound. ⁇
  • FIG. 25 is a schematic diagram showing an example of the structure of a ribosome to which 3, -sialylratosatosamine trisaccharide is bound.
  • FIG. 26 is a schematic diagram showing an example of the structure of a ribosome to which a 6, -sialylratatosamine trisaccharide is bound.
  • FIG. 27 is a graph showing the amount of the four ribosome complexes transferred into the blood 10 minutes after intestinal administration.
  • Figure 28 shows the amount of the four ribosome complexes transferred into the blood 10 minutes after intestinal administration.
  • FIG. 29 is a graph showing the amount of the four ribosome complexes transferred into the blood 10 minutes after intestinal administration. '
  • FIG. 30 is a graph showing the amount of the four ribosome complexes transferred into the blood 10 minutes after intestinal administration.
  • -FIG. 31 is a diagram showing the amount of the four ribosome complexes transferred into the blood 10 minutes after intestinal administration.
  • FIG. 32 is a schematic diagram of a liposome bound with tris (hydroxymethyl) aminomethane as a comparative sample.
  • FIG. 33 is a schematic diagram showing a structural example of a ribosome to which a Lewis X-type trisaccharide is bound.
  • FIG. 34 is a schematic diagram showing an example of the structure of a ribosome to which a sialyl Lewis X-type quail bran chain is bound. ' s
  • FIG. 35 is a schematic diagram showing an example of the structure of a ribosome modified by ratatosuni sugar chain.
  • FIG. 36 is a schematic diagram showing an example of the structure of a ribosome modified by 2′-fucosyllatases trisaccharide.
  • FIG. 37 is a schematic diagram showing a structural example of a ribosome modified with difucosyl ratatose tetrasaccharide.
  • FIG. 38 is a schematic diagram showing a structural example of a ribosome modified with a 3-fucosyl lactose trisaccharide.
  • FIG. 39 is a diagram showing the anticancer effect in tumor-bearing mice by intravenous tail injection of ribosomes linked to alpha 1,6 mannobiose disaccharide.
  • FIG. 40 is a photograph showing the carcinostatic effect of a liposome to which ⁇ 1,6 mannobiose disaccharide is bound by intravenous tail injection on a tumor-bearing mouse.
  • FIG. 41 is a diagram showing the anticancer effect in tumor-bearing mice by oral administration of ribosomes linked with 3-fucosyllactose trisaccharide.
  • FIG. 42 is a photograph showing the antitumor effect of a liposome bound with 3-fucosyllactose trisaccharide on oral administration to a tumor-bearing mouse.
  • FIG. 43 is a graph showing the results of a test for enhancing the cytotoxicity of L-Dox and L-Dox-SLX by interferon ⁇ .
  • FIG. 44 is a diagram showing the results of a test for suppressing the cytotoxicity of L-Dox-SLX when adding IFN using an L-selectin neutralizing antibody.
  • FIG. 45 is a graph showing time-dependent changes in blood doxorubicin concentration in tumor-bearing mice.
  • FIG. 46 is a graph showing the time-dependent change in the concentration of doxorubicin in the tumor of the tumor-bearing mouse.
  • FIG. 47 is a photomicrograph showing the distribution of doxorubicin in tumor tissues and cells with a small amount of tumor.
  • FIG. 48 is a graph showing the anti-cancer effect in tumor-bearing mice by intravenous tail injection.
  • FIG. 49 is a photograph showing the anticancer effect of a tumor-bearing mouse by intravenous tail injection.
  • FIG. 50 is a diagram showing the tumor weight of each group showing the anti-cancer effect in the tumor-bearing mouse by intravenous tail injection.
  • FIG. 51 is a diagram showing a comparison result of blood retention of ribosomes subjected to two types of hydrophilization treatment and untreated ribosomes 5 minutes after intravenous injection into a cancer-bearing mouse tail.
  • FIG. 52 is a photograph of the limb of an inflamed RA mouse.
  • FIG. 53 is a diagram showing the results of treatment of RA mice by intravenous administration of prednisolone-embedded DDS.
  • FIG. 54 shows the results of treatment of RA mice by oral administration of prednisolone-embedded DDS.
  • FIG. 55 shows the results of treatment by administering an appropriate amount of prednisolone to RA mice.
  • the present invention relates to a targeting ribosome having various sugar chains on its surface and a ribosome controlling intestinal absorption.
  • target directivity refers to the specific reach of a specific tissue or organ or a target site such as a disease site such as cancer when administered into a living body.
  • the intestinal absorption controllability refers to the property of being taken up into a living body through the intestinal tract, that is, the property of being able to control the rate and extent of intestinal absorption, which is intestinal absorption.
  • the ribosome usually means a closed vesicle composed of a lipid layer and a water layer inside the membrane layer.
  • the ribosome of the present invention has a sugar chain bonded to its surface, that is, a lipid layer. It may be bonded to a lipid layer or may be covalently bonded via a linker protein which is a biological protein containing a human-derived protein such as human serum albumin.
  • the sugar chains are linked by controlling their type and density.
  • sugar chains can be used depending on the target tissue, organ, cancer or the like of the intestinal absorption control ribosome of the present invention and the target liposome of the present invention. It is known that E-selectin and P-selectin expressed on the vascular endothelial cells at the lesion and sialyl Lewis X, a sugar chain expressed on the cell membrane of leukocytes, are strongly bound ⁇ .
  • This ribosome has a sugar chain capable of reacting with this sialyl Lewis X sugar chain or a protein having a lectin-like sugar chain binding site such as E-selectin or P-selectin.
  • vascular endothelial cells will specifically accumulate at the lesion site of cancers expressing E-selectin, P-selectin, etc.
  • sites that express E-selectin, P-selectin, etc. are sites where inflammation or angiogenesis occurs, and the blood vessels in such sites have enlarged intercellular spaces of endothelial cells, It is thought that the ribosomes spread from the gap to the lesion site and its surroundings. The diffused ribosome is taken up (endocytosis) by the cells at and around the lesion site, releasing the drug contained inside the cells. By such a mechanism, it is effective against inflammatory diseases or cancer.
  • sugar chain to be bound to the ribosome of the present invention examples include a sugar chain capable of reacting with a protein having a lectin-like sugar chain binding site such as E-selectin and P-selectin as described above.
  • E-selectin, P-selectin, etc. mean selectin, DC-SIGN, DC-SGNR, collectin, C-type lectin such as mannose binding protein, I type lectin such as Siglec, mannose-6-phosphate receptor P-type body It refers to various lectins (sugar chain recognition proteins) such as lectin, R-type lectin, L-type lectin, M-type lectin and galectin.
  • intestinal absorption-controlling ribosomes include 3'-sialyl lactose trisaccharide (the structural formula is shown in Fig. 23; the same applies hereinafter), 6'-sialyl lactose trisaccharide (Fig. 24), 3'-sialyl lactose trisamine sugar ( Figure 25) and 6 '- Shiarirurata Tosamin sugar chains (Fig.
  • alpha 1,2 mannobiose two sugar chain as targeting ribosomes
  • Figure 1 alpha 1, 3 mannobiose disaccharide
  • Figure 2 Alqua 1,4 mannobiose disaccharide
  • Fig. 3 alpha 1,6 mannobiose disaccharide
  • Fig. 4 alpha 1,3 alpha 1,6 manno triose trisaccharide
  • oligo Oligomannose 3 pentasaccharide Fig. 6
  • Oligomannose 4b hexasaccharide Fig. 7
  • Oligomannose 5 heptasaccharide Fig. 8
  • Oligomannose 6 octasaccharide Fig.
  • the ribosome used in the present invention is to improve the stability of the membrane, to prevent leakage of the drug or gene to be encapsulated, to enable the membrane surface to be subjected to hydrophilic treatment, and to convert proteins at various densities.
  • we will make various efforts such as the following lipids and glycolipids as the constituent components. Was prepared.
  • Examples of the lipid constituting the ribosome of the present invention include phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, long-chain alkyl phosphates, dicetyl phosphates, gandariosides, glycolipids, Phosphatidyldali serols, sphingomyelins, cholesterols, etc., and phosphatidylcholines include dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, and the like.
  • phosphatidylethanolamines include dimyristoylphosphatidylethanolamine, dipalmitoinolephosphatidinolethananolamine, distearoylphosphatidylethanolamine, and the like, phosphatidic acids or Examples of long-chain alkyl phosphates include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, and dicetyl phosphate.
  • Gandariosides include gandarioside GM1, gandarioside GDla, Gandarioside GT 1b, etc .; glycolipids: galactosyl ceramide, darcosyl ceramide, ratatosyl ceramide, phosphatide, gloposide, etc .; phosphatidylglycerols: dimyristoyl sulfide Staple ⁇ Ji Jill glycerol, Gino Noremi Toys Honoré phosphatidyl
  • lipid constituting the ribosome of the present invention phosphatidylcholines (molar ratio 0 to 70%), phosphatidylethanolamines (molar ratio 0 to 30%), phosphatidic acids, and long-chain alkyls
  • lipids selected from the group consisting of phosphates and dicetyl phosphates (molar ratio 0-30%), gandariosides, glycolipids, phosphatidylglycerols and sphingomyelins And those containing one or more lipids (molar ratio 0 to 40%) selected from cholesterols (molar ratio 0 to 70%).
  • the ribosome itself can be produced according to a well-known method, and examples thereof include a thin film method, a reverse layer evaporation method, an ethanol injection method, and a dehydration-rehydration method.
  • the method for producing the ribosome of the present invention is as follows. Or a mixture of a lipid containing phosphatidyl glycerol and a surfactant sodium cholate Prepare micelles.
  • the blending of long-chain alkyl phosphates such as phosphatidic acid or dicetyl phosphate is essential for negatively charging liposomes, and the blending of phosphatidylethanolamines is not essential.
  • Mixing of gangliosides or glycolipids or phosphatidylglycerols as the hydrophilization reaction site is indispensable as the binding site of the linker protein.
  • At least one lipid selected from the group consisting of gandariosides, glycolipids, phosphatidylglycerols, sphingomyelins, and cholesterols assembles in the ribosome and binds a linker-protein scaffold (raft). ).
  • the ribosome of the present invention is further stabilized by forming a raft capable of binding such a protein. That is, the ribosome of the present invention comprises at least one kind of lipid selected from the group consisting of ganglioside, glycolipid, phosphatidylglycerols, sphingomyelins, and cholesterols for binding a linker protein. It contains ribosomes with ft.
  • a liposome is produced by performing ultrafiltration of the mixed micelle obtained thereby.
  • the ribosome used in the present invention may be an ordinary ribosome, but its surface is desirably made hydrophilic. After the ribosome is prepared as described above, the ribosome surface is made hydrophilic. The ribosome surface is made hydrophilic by binding a hydrophilic compound to the liposome surface.
  • a low-molecular-weight hydrophilic compound preferably a low-molecular-weight hydrophilic compound having at least one 0H group, and more preferably a low-molecular-weight hydrophilic compound having at least two 0H groups Compounds.
  • a low-molecular-weight hydrophilic compound having at least one amino group that is, a hydrophilic compound having at least one 0H group and at least one amino group in a molecule is also exemplified. Since the hydrophilic compound is a small molecule, it does not easily cause steric hindrance to the bran chain and does not hinder the progress of the sugar chain molecule recognition reaction by the lectin on the target cell membrane surface. In addition, the hydrophilic compound does not include a sugar chain to which lectin used for directing a specific target such as lectin in the sugar chain-modified liposome of the present invention can bind.
  • Tris (hydroxyalkyl) amino alcohols such as aminoalkanes, including tris (hydroxymethyl) aminoalkane, and the like. More specifically, tris (hydroxymethylinole) aminoethane, tris (hydroxylethylamino) ethane, and the like. Tris (hydroxypropyl) aminoethane, tris (hydroxymethyl) aminoamino, tris (hydroxyxethyl) aminomethane, squirrel (hydroxypropyl) aminomethane, tris (hydroxymethyl) aminopropane, tris (hydroxyxethyl) aminopropane, tris (Hydroxypropyaminopropane, etc.
  • a compound in which an amino group is introduced into a low molecular weight compound having a 0-group can also be used as the hydrophilic compound of the present invention.
  • examples include, but are not limited to, compounds in which an amis group has been introduced into a bran chain to which lectin does not bind, such as cellobiose, etc.
  • a bivalent reagent for cross-linking on the phosphatidyl ethanolamine of the ribosome membrane and tris (Hydroxymethyl) Aminomethane is used to make the ribosome surface hydrophilic.
  • One formula of a hydrophilic compound is represented by the following formulas (1), (2), and (3).
  • R1, R3 and R5 represent a linear or branched hydrocarbon chain of C1 to C40, preferably C1 to C20, more preferably C1 to C10, and R2, R4 are absent or C1 to C40, preferably C1 to C20, more preferably to
  • X represents a reactive functional group that binds to ribosomal lipids directly or with a bivalent bridging reagent, for example, C00H, NH, thigh. , CH0,
  • n indicates a natural number.
  • the surface of the liposome hydrophilized with such a hydrophilic compound is thinly covered with the hydrophilic compound. However, since the thickness of the cover of the hydrophilic compound is small, even when a sugar chain is bound to the liposome, the reactivity of the bran chain or the like is not suppressed.
  • Ribosome hydrophilization can be performed by a conventionally known method, for example, polyethylene dalicol, It can also be carried out by adopting a method such as a method of producing ribosomes using a phospholipid in which a polyvier alcohol, a maleic anhydride copolymer or the like is covalently bonded (JP-A-2000-302685). be able to.
  • the ribosome surface hydrophilic using tris (hydroxymethyl) aminomethane.
  • tris (hydroxymethyl) amino methane is preferable in several points as compared with the conventional method for hydrophilization using polyethylene glycol and the like.
  • tris (hydroxymethyl) aminomethane is a low-molecular-weight substance, and thus has a high molecular weight such as conventional polyethylene glycol.
  • it is particularly preferable because it does not hinder the steric hindrance to the sugar chain and does not hinder the progress of the sugar chain molecule recognition reaction by lectin (sugar chain recognition protein) on the target cell membrane surface.
  • the liposome according to the present invention has good particle size distribution, component composition, and dispersion characteristics even after the hydrophilization treatment, and also has excellent long-term storage stability and in vivo stability. Preferred for use.
  • a ribosome solution obtained by a conventional method can be used to prepare bisulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobissuccinimidyl propionate.
  • the divalent reagent is added to and allowed to react with the divalent reagent, thereby binding the divalent reagent to lipids such as dipalmitoylphosphatidylethanolamine on the ribosome membrane, and then tris (hydroxymethyl) aminoaminomethane is added to one of the divalent reagents.
  • ribosomes obtained by hydrophilizing ribosomes are extremely stable in vivo, and have a long half-life in vivo even without the attachment of target-directed sugar chains as described below. It can be suitably used as a drug carrier in a drug livery system.
  • the present invention also includes a liposome whose surface is made hydrophilic with a low molecular compound. .
  • the present invention also includes ribosomes that are not bound to sugar chains that have been hydrophilized using the above-mentioned hydrophilizing compound.
  • Such hydrophilized ribosomes have the advantage that the stability of the liposome itself is increased and that the recognition of sugar chains is enhanced when sugar chains are bound.
  • the constituent lipids of the ribosome are phosphatidylcholines (molar ratio 0 to 70%), phosphatidylethanolamines (molar ratio 0 to 30%), phosphatidic acids, One or more lipids (molar ratio 0-30%) selected from the group consisting of dialkyl alkyl phosphates and dicetyl phosphates, cancer! G, liosides, glycolipids, phosphatidylglycerols and sphingomyelins, one or more lipids (molar ratio 0 to 40%), and cholesterols ('molar ratio 0 to 40%). (70%).
  • the present invention further includes a method for making the liposome hydrophilic by binding the above-mentioned hydrophilizing compound to the ribosome.
  • the term also includes liposomes that have been made hydrophilic and have no sugar chain attached. By binding a bran chain to a ribosome to which no sugar chain is bound, the targeting ribosome or intestinal absorptive ribosome of the present invention can be produced.
  • any of the above sugar chains may be directly bound to the ribosome prepared as described above, or a bran chain may be bound via a linker protein.
  • the type of sugar chain to be bound to the ribosome is not limited to one type, and a plurality of sugar chains may be bound.
  • the plurality of sugar chains may be a plurality of sugar chains having a binding activity to different lectins commonly present on the cell surface of the same tissue or organ, or may be present on the cell surface of a different tissue or organ. May be sugar chains having binding activity to different lectins.
  • glycans as in the former, it is possible to reliably target a specific target tissue or organ, By selecting a plurality of sugar chains as in the case of one, a plurality of targets can be directed to one type of liposome, and a multipurpose target-directed ribosome can be obtained.
  • a protein derived from a living organism particularly a human-derived protein
  • the protein derived from a living body is not limited, and examples thereof include proteins present in blood such as albumin, and other physiologically active substances present in a living body.
  • serum albumin of animals such as human serum albumin (HSA) and human serum albumin (BSA) can be mentioned.
  • HSA human serum albumin
  • BSA human serum albumin
  • the liposome of the present invention is very stable, and can be subjected to post-processing such as protein binding, linker protein binding, and sugar chain binding after liposome formation. Therefore, it is possible to produce various ribosomes according to the purpose by combining a different protein or a linker protein or a sugar chain according to the purpose after producing a large amount of ribosomes. It is.
  • a sugar chain is directly bound to a lipid constituting a liposome via a linker protein.
  • the ribosome of the present invention has complex carbohydrate-type ligands such as glycolipids and glycoproteins, and has been hydrophilized with a low-molecular compound. Posome.
  • the ribosome when the targeting ribosome of the present invention is used as a medicine, the ribosome needs to contain a compound having a pharmaceutical effect.
  • the compound having a medicinal effect may be a compound capable of being encapsulated in ribosomes or bound to the ribosome surface, but a protein having a medicinal effect may be used as the linker protein.
  • the protein may also serve as a linker protein for binding ribosomes to sugar chains and a protein having a medicinal effect.
  • the protein having a medicinal effect include bioactive proteins.
  • a protein is bound to the ribosome surface. Liposomes, was treated with N a I 0 4, P b (O 2 CCH 3) 4, N a B i 0 oxidant such as 3, oxidizing the gangliosides present on ribosomes film surface, then, N a BH 3 CN, using a reagent such as N a BH 4, a ganglioside on linker protein and Liposomes Ichimu film surface, is bound Ri by the reductive Amino reaction. It is preferable that the linker protein is also made hydrophilic, and a compound having a hydroxy group is bound to the linker protein.
  • the compound used for the above-mentioned hydrophilization such as tris (hydroxymethyl) aminomethane may be bound to the linker protein on the ribosome by using a bivalent reagent such as norrescine.
  • a crosslinking bivalent reagent is bonded to all amino groups of the linker protein. Then, a glycosylamine compound obtained by reacting the reducing end of each type of sugar chain with glycosylamination is prepared, and the amino group of this sugar chain is linked to the above-mentioned crosslinked divalent reagent on the liposome. Binds some other unreacted ends.
  • the covalent bond between the sugar chain and / or hydrophilic compound and the ribosome, or the covalent bond between the sugar chain and the Z or hydrophilic compound and the linker protein, is caused by the intracellular liposome. It is also possible to cut when taken in. For example, when a linker protein and a sugar chain are covalently linked via a disulfide bond, the sugar chain is cleaved by reduction in the cell. When the sugar chain is cleaved, the surface of the liposome becomes hydrophobic, and the liposome binds to the biological membrane, disrupting the membrane stability and releasing the drug contained in the ribosome.
  • the sugar chain is not bound to the surface of the protein on the surface of the sugar chain-bound ribosome membrane obtained in this manner.
  • Perform processing In other words, the unreacted end of the bivalent reagent bound to the protein on the ribosome is subjected to a binding reaction with the above-mentioned compound used for hydrophilization such as tris (hydroxymethyl) aminoaminomethane, and the entire liposome surface is hydrophilized. I do.
  • Hydrophilization of the linker protein on the liposome surface improves the transferability of various tissues, the retention in blood and the transferability to various tissues. This is because the surface of the liposome and the surface of the linker protein are made hydrophilic, so that the parts other than the sugar chain appear in each tissue as if they were water in the living body. This is probably due to the fact that only the sugar chain is recognized by the lectin (sugar chain recognition protein) of the target tissue without being recognized by the tissue.
  • the sugar chain is bound to the linker protein on the ribosome.
  • the reducing end of the saccharide constituting the sugar chain is glycosylated with an ammonium salt such as NH 4 HC ⁇ 3 or NH 2 COONH 4 , followed by bissulfosuccinimidyl verate, Disuccinimidyl glutarate, dithiobissuccinimiderup mouth pionate, disuccinimidyl suberate, 3,3'-dithiobissunorrefosucciniminoleproionate, ethylene glycol Using a bivalent reagent such as cineate or ethylene glycol bissulfostasimidyl succinate, the linker protein bound on the liposome membrane surface and the daricosylamino sugar are bound to each other.
  • the liposomes as shown in 2, 23-26 and 33-38 are obtained.
  • the liposome of the present invention the particle size of the liposome such as a sugar chain is 30 to 500 nm, preferably
  • the zeta potential of the ribosome surface of the present invention In 37. In C, it is ⁇ 50 to 10 mV, preferably ⁇ 40 to 0 mV, and more preferably ⁇ 30 to ⁇ 10 mV.
  • the binding density of the sugar chain in the case where the sugar chain is linked is 1 to 60, preferably 1 to 40, and more preferably 1 to 20 per one molecule of the linker protein bound to the ribosome.
  • per 1 liposome particle when using a linker protein, 1 to 30,000, preferably 1 to 20,000, more preferably 1 to 10,000, or 100 to 30,000, preferably 100 to 20,000, More preferably 100 to: 10,000, or 500 to 30,000, preferably 500 to 20,000, more preferably 500 to 10,000.
  • a maximum of 1 to 500,000, preferably 1 to 300,000, more preferably 1 to 100,000 or more sugar chains can be bound per ribosome particle.
  • directivity to each target cell or tissue can be controlled by variously selecting the structure of the sugar chain and the amount of sugar chain to be used.
  • the lectin to be specifically bound is determined by the type of sugar chain and the amount of sugar chain bound, and specifically reaches a specific tissue or organ. Further, by selecting the sugar chain structure and the sugar chain binding amount, it is possible to reach a disease site such as a cancer tissue.
  • directivity to each target cell or tissue can be controlled by variously selecting the structure of the sugar chain and the amount of sugar chain to be used.
  • the liposome of the present invention can be used in the blood, liver, spleen, lung, brain, small intestine, heart, thymus, kidney, knee, muscle, large intestine, bone, bone marrow, cancer tissue, inflamed tissue, lymph node, etc. Orient the tissue or organ. For example, as shown in FIGS.
  • alpha 1,2 mannobiose disaccharide alpha 1,3 mannobiose disaccharide, alpha 1,4 Mannobiose disaccharide, alpha 1,6 Mannobiose 2
  • Sugar chains alpha 1, 3 alpha 1, 6 mannotriose trisaccharide, oligomannose 3 pentasaccharide, oligomannose 4 b hexasaccharide, oligomannose 5 heptasaccharide, oligomannose 6 octasaccharide, oligomacinose 7
  • Oligomannose 9 ""-oligomannose 9 "” all ribosomes linked to sugar chains have high directivity to blood, lung, brain, cancer tissues, heart and small intestine.
  • ribosomes linked with alpha 1,2 mannobiose disaccharide, oligomannose 35 pentasaccharide, and oligomannose 4b hexasaccharide have high directivity to the liver.
  • ribosomes linked to alpha 1,3 'mannobiose disaccharide, alpha 1,3 mannobiose sugar chain, and alpha 1,3 alpha 1,6 mannotriose trisaccharide are transferred to the spleen. High orientation.
  • alpha 1, 2 Man'nobionisu disaccharide as shown FIG.
  • the ribosomes shown in FIGS. 23 to 26 of the present invention generally have very high intestinal absorption, but furthermore, by controlling the sugar chain density on the ribosome, the intestinal absorption is improved. It is possible to control and transfer the drug to the target site more efficiently, and to reduce side effects.
  • FIGS. 27 to 30 in the examples the translocation from the intestinal tract to the blood (that is, intestinal absorptivity) when the amount of sugar chain binding in the four types of sugar chain-modified ribosomes is changed in three stages is shown. Is shown.
  • the sugar chain binding amount of change Gyotsu by allowed to bind in a concentration of three stages of sugar chains the linker one protein binding ribosomes (l) 50 ig, 2) 200 ⁇ ⁇ , 3) lmg) It is something.
  • the intestinal absorbability gradually decreases.
  • 3,3-sialyl lactosamine sugar chains intestinal absorbability is increased.
  • the ribosome By including a compound having a 'pharmaceutical effect' in the ribosome of the present invention, the ribosome reaches the tissue or organ of interest, the liposome is taken up by cells of the tissue or organ, and releases a compound having a medicinal effect, thereby exhibiting a medicinal effect.
  • the ribosome In the case of a sugar chain-linked ribosome, the ribosome reaches a specific tissue or organ due to the targeting property of the ⁇ chain.
  • liposomes are very stable in the body, have a long half-life in the body, and can reach specific tissues or organs.
  • the compound having a pharmaceutical effect is not limited, and known proteins and known pharmaceutical compounds can be widely used.
  • the ribosome of the present invention can be used as a therapeutic agent for a specific disease by including a pharmaceutical compound for a specific disease such as an anticancer agent.
  • the compound having a medicinal effect included in the ribosome of the present invention includes DNA, RNA, and siRNA for gene therapy.
  • Pharmaceutical compounds contained in the ribosome of the present invention include alkylating anticancer agents, metabolic antagonists, plant-derived anticancer agents, anticancer antibiotics, BRM * cytokines, platinum complex anticancer agents, immunotherapeutic agents, and hormonal agents.
  • Tumor drugs such as anticancer drugs, monoclonal antibodies, drugs for central nervous system, drugs for peripheral nervous system and sensory organs, drugs for respiratory diseases, drugs for circulatory organs, drugs for digestive organs, drugs for hormonal systems, drugs for urology and reproductive organs, Topical drugs, vitamins, nourishing tonics, drugs for blood and body fluids, metabolic drugs, antibiotics, chemotherapy drugs, testing drugs, anti-inflammatory drugs, eye disease drugs, central nervous system drugs, autoimmune drugs, cardiovascular Drug, diabetes.
  • Drugs for lifestyle-related diseases such as hyperlipidemia or various drugs for oral, pulmonary, transdermal or transmucosal, corticosteroids, immunosuppressants, antibacterials, antivirals, angiogenesis inhibitors, cytokine chemokines , Anti-cytokine antibodies, anti-chemokine antibodies, anti-cytokine and chemokine receptor antibodies, siRNA, miRNA, smRNA, antisense, nucleic acid preparations related to gene therapy such as 0DN and DNA, neuroprotective factors, various antibody drugs, etc. .
  • alkylating agents such as nitrogen mustard hydrochloride-N-oxide, cyclophosphamide, diphosphamide, brusfan, dimustine hydrochloride, mitbronitol, menolephalan, dacanolepazine, ranimustine, estramustine sodium phosphate, etc.
  • the ribosome of the present invention can be used for treating diseases such as cancer and inflammation.
  • cancer includes all neoplastic diseases such as tumors and leukemias.
  • the sugar chain-modified ribosome of the present invention is administered with these drugs contained, the drugs accumulate at cancer or inflammation sites as compared with the case where the drugs are administered alone. Compared with the case of single administration, it can accumulate 2 times or more, preferably 5 times or more, more preferably 10 times or more, particularly preferably 50 times or more.
  • the compound having a medicinal effect may be encapsulated in ribosomes or bound to the liposome surface.
  • a protein can be bound to the surface by the same method as the linker-protein binding method described above, and other compounds can be bound by a known method by utilizing a functional group of the compound. It can be done.
  • the encapsulation in the ribosome is performed by the following method.
  • Well-known methods may be used to encapsulate drugs and the like into ribosomes. For example, a solution containing a drug and the like and phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids or long-chain alkyl phosphates are used.
  • the ribosome preparation obtained by encapsulating a drug or a gene which can be used for treatment or diagnosis into the ribosome of the present invention can selectively control the translocation to cancer tissues, inflammatory tissues and various tissues. It is intended to enhance the efficacy of the therapeutic drug or diagnostic agent by concentrating it on target cells or tissues, or to reduce side effects by reducing the uptake of the drug into other cells or tissues.
  • the ribosome or sugar chain-modified ribosome of the present invention can be administered in various forms as a pharmaceutical composition.
  • administration forms include ophthalmic administration with eye drops, etc., oral administration with tablets, capsules, granules, powders, syrups, etc., and parenteral administration with injections, drops, suppositories, etc. be able to.
  • Such a composition is produced by a known method and includes a carrier, a diluent, and an excipient that are commonly used in the field of formulation. For example, gelling agents, lactose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • the injection is prepared by dissolving, suspending or emulsifying the sugar chain-linked ribosome of the present invention in a sterile aqueous or oily liquid commonly used for injections.
  • Physiological saline solution, isotonic solution containing glucose and other adjuvants, etc. are used as aqueous liquids for injection, and suitable solubilizing agents, for example, alcohols, polyalcohols such as propylene daricol, and nonionic It may be used in combination with a surfactant or the like.
  • suitable solubilizing agents for example, alcohols, polyalcohols such as propylene daricol, and nonionic It may be used in combination with a surfactant or the like.
  • Sesame oil, soybean oil, and the like are used as the oily liquid, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent.
  • the route of administration of the pharmaceutical composition of the present invention is not limited, and includes eye drops, oral administration, intravenous injection, intramuscular injection and the like.
  • the dose can be appropriately determined depending on the severity of the disease and the like, but a pharmaceutically effective amount of the composition of the present invention is administered to the patient.
  • “administering a pharmaceutically effective amount” refers to administering to a patient a drug at an appropriate level for treating various diseases.
  • the frequency of administration of the pharmaceutical composition of the present invention is appropriately selected according to the patient's condition. Ribosomes with glycosides on the surface are suitable for parenteral (intravenous) and oral administration. Dosage is several minutes less than conventional non-targeted ribosomes.
  • the amount of drug contained in the ribosome per kg of body weight is 0.0001 to 1000 mg, preferably 0.0001 to 10 mg, and more preferably 0.0001 to 1000 mg.
  • the ribosome of the present invention does not contain a sugar chain having a targeting property. It also includes a ribosome to which a hydrophilic compound is bound, but the ribosome also has a high stability in vivo and a long half-life due to the hydrophilic treatment, and thus has a sufficient effect at a low dose. '
  • a labeled compound such as a fluorescent dye or a radioactive compound is bound to the ribosome.
  • the labeled compound-bound ribosome binds to the diseased site, the labeled compound is taken up by the diseased cells, and the disease can be detected and diagnosed using the presence of the labeled compound as an index.
  • a cosmetic or a cosmetic product encapsulated in or bound to the ribosome of the present invention can be used as a cosmetic composition.
  • cosmetics generally refer to ⁇ rubbing, dusting, and the like on a body to clean, beautify, increase its appeal, change its appearance, or keep its skin or hair-hair healthy, That are intended to be used in such a way that they have a modest effect on the human body.
  • cosmetics refers to the above-mentioned general cosmetics as well as “purposes of use that are mild in use, do not use for the treatment or prevention of diseases, and affect the structure and function of the body. Without quasi-drugs ". Cosmetics include, for example, those that act on cells such as the skin to activate the cells.
  • Cosmetics include cosmetics for skin, hair, hair and scalp.
  • skin bleaching agents such as magnesium phosphate-ascorbate, kojic acid, plancentax, arbutin, and ellagic acid
  • vitamins such as vitamin A, vitamin B, vitamin C, and vitamin E
  • Hormones such as estrogen, estradiol, estrone, ethurestradione, conoretisone, hydroconoretisone, predison, prednisolone, citrate, tartaric acid, lactic acid, aluminum chloride, aluminum chloride, potassium aluminum sulfate, myopan, allantrin
  • Skin astringents such as chlorhydroxyaluminum, allantoindihydroxyaluminum, zinc parafufenol sulfonate, and zinc sulfate, cantharistinus, capsicum tincture, gingerbread tincture, sempuri extract, garlic extract, hinoki Thiol, Karupuroniumu chloride, hair growth promoting agents such as pentadecanoic acid
  • composition of the present invention in which cosmetics are encapsulated or bound to ribosomes remains on the skin surface, where the cosmetics contained in the ribosomes are released and exert their effects on the skin surface.
  • the ribosome is absorbed percutaneously and reaches the stratum corneum or the tissue below the stratum corneum.
  • the ribosome is taken up by the cells of the tissue, releasing cosmetics and exerting a medicinal effect.
  • the liposome used in the cosmetic composition may not have a sugar chain attached, in which case the liposome stays in the skin or the tissue under the skin, where it is made up.
  • a sugar chain may be bound so as to target the inflamed part of the skin and specifically accumulate in the inflamed part.
  • the cosmetic composition of the present invention may contain, in addition to the above-mentioned ribosome, an aqueous component, an oily component, and the like which are usually blended as a cosmetic.
  • the aqueous component include humectants, thickeners, alcohols, and the like.
  • the humectants include glycerin, propylene glycol, and polyhydric alcohols.
  • the thickeners include tragacanth gum, pectin, and alginic acid. Salts and the like, and alcohols include ethanol, isopropyl alcohol and the like.
  • Oily components include olive oil, camellia oil, castor oil, wax, oleic acid, solid paraffin, ceresin, wax, petrolatum, liquid paraffin, silicone oil, synthetic ester oil, synthetic polyether, and the like.
  • a food in which a functional food, a dietary supplement or a health supplement is encapsulated or bound to the ribosome of the present invention can be used.
  • the functional food, nutritional supplement or health supplement that can be used in the present invention is not limited, and includes any food that has been designed so as to be ingested to effectively exert the food function and processed and converted.
  • ribosomes that can be used in the present invention, such as carrots, carrots, taresone, fermented plant foods, DHA, EPA, ARA, kelp, cabbage, aloe, megsulino tree, hops, oyster extract, picgenol, sesame, etc. Or it can be exemplified as a health supplement.
  • These may be contained in the ribosome as it is, or may contain a processed product such as an extract.
  • Food-food compositions containing liposonym are taken orally.
  • the ribosome to be used may not have a sugar chain attached thereto, and may have a sugar chain that enhances intestinal absorption or a sugar chain targeting a specific tissue or organ bound.
  • the ribosome of the present invention When used as a food composition, it may be processed into a food such as a liquid beverage, a gel food, or a solid food. Further, it may be processed into tablets, granules and the like.
  • the food composition of the present invention can be used as a functional food, dietary supplement or health supplement depending on the type of food contained in the ribosome.
  • ribosomes containing DHA can be used as functional foods, dietary supplements or health supplements that are effective in mild senile dementia and memory improvement.
  • Ribosomes are based on the method of Saikyo (Yamazaki, N., Kodama, M. and Gabius, H.-J. (1994)
  • the obtained lipid membrane is suspended in 3 ml of TAPS buffer (pH 8.4), sonicated, and cleared. A micellar suspension was obtained. Further, the micelle suspension was subjected to ultrafiltration using a PM10 membrane (Amicon Co., USA) and a PBS buffer (pH 7.2) to prepare 10 ml of uniform ribosomes (average particle diameter 100 nm). '
  • this ribosome solution was subjected to ultrafiltration using an XM300 membrane and a CBS buffer (pH 8.5).
  • CBS buffer night (pH 8.5) 1 ml
  • the binding reaction between BS3 bound to the lunar substance on the liposome membrane and tris (hydroxymethyl) aminomethane was completed.
  • the hydroxyl group of tris (hydroxymethyl) aminomethane was placed on the lipid dipalmitoylphosphatidinolethananolamine of the ribosome membrane to make it hydrated and hydrophilized.
  • This ribosome solution was added to 20mg of human serum albumin (HSA) was stirred for 2 hours at 25 ° C, then with PBS (P H 8. 0) in 2M NaB CN 100 ⁇ 1 were added 10 ° C The HSA was bound by agitation with a coupling reaction between the ganglioside on the ribosome and HSA. Then, after ultrafiltration with an XM300 membrane and a CBS buffer (pH 8.5), 10 ml of an HSA-bound ribosome solution was obtained.
  • HSA human serum albumin
  • Example 4 Alpha 1, 2 on ribosome membrane-bound human serum albumin (HSA) Mannobiose binding of two bran chains.
  • HSA human serum albumin
  • Alpha 1, 2 mannobiose disaccharide (Calbiochem Co., USA) 50 g was added to 0. 5 ml aqueous solution prepared by dissolving NH 4 HC0 3 of 0. 25 g, After stirring for 3 days at 37 ° C, 0. 45 ⁇ Filtration through a filter of ⁇ completed the amination reaction of the reducing end of the sugar chain to obtain 50 ⁇ g of a glycosylamine compound of an alpha 1,2 mannobiose disaccharide chain.
  • Anorefa 1,3 mannobiose disaccharide (Calbiochem Co., USA) 50 g was added to 0. 5 ml aqueous solution prepared by dissolving NH 4 HC0 3 of 0. 25 g, After stirring for 3 days at 37 ° C, 0. 45 ⁇ Filtration with a ⁇ filter completed the amination reaction of the reducing end of the sugar chain to obtain 50 g of a glycosylamine compound of an alpha 1,3 mannobiose disaccharide.
  • Anorefa 1,4 mannobiose disaccharide (Calbiochem Co., USA) 50 ⁇ g was added to 0. 5 ml aqueous solution prepared by dissolving NH 4 HC0 3 of 0. 25 g, After stirring for 3 days at 37 ° C, 0. 45 Filtration through a ⁇ m filter completed the amination reaction of the reducing end of the sugar chain to obtain 50 ⁇ g of a glycosylamine compound of an alpha 1,4 mannobiose disaccharide chain.
  • Alpha 1, 6 mannobiose disaccharide (Calbiochem Co., USA) 50 g was added to 0. 5 ml aqueous solution prepared by dissolving NH 4 HC0 3 of 0. 25 g, After stirring for 3 days at 37 ° C, 0. 45 111 Then, the amination reaction at the reducing end of the sugar chain was completed to obtain 50 ig of a glycosylamine compound of an alpha 1,6 mannobiose disaccharide chain.
  • a liposome (abbreviation: A6) 2 ⁇ (total lipid amount 2 mg, total protein amount 200 g, average particle size) in which alpha 1,6 mannobiose disaccharide, human serum albumin and liposome were combined as shown in Fig. 4 'lOOnm).
  • Alpha 1, 3 alpha 1, 6 mannose triose trisaccharide (Calbiochem Co., USA) and 50 / g was added to 0. 5 ml aqueous solution dissolving N3 ⁇ 4HC0 3 of 0. 25 g, After stirring for 3 days at 37 ° C, The solution was filtered through a 0.45 ⁇ filter to complete the amination reaction of the reducing end of the sugar chain, thereby obtaining 50 ⁇ g of a glycosylamine compound of an alpha 1,3 alpha 1,6 mannotriose trisaccharide.
  • Example 9 Binding of oligomannose tripentasaccharide to liposomal membrane-bound human serum albumin (HSA) '' Oligomannose 3 Gotokusari (Calbiochem Co., USA) 50 g was added to NH 4 HC0 3 of 0. 25 g to 0. 5 ml aqueous solution Dissolve After stirring 3 days at 37 ° C, 0. of 45 m After filtration through a filter, the amination of the reducing end of the sugar chain was completed to obtain 50 ⁇ g of a glycosylamine compound of oligomannose 35 pentasaccharide.
  • HSA liposomal membrane-bound human serum albumin
  • Example 3 1 mg of the liposome solution obtained in Example 3 was added to 5 ml of the liposome solution, and 3 mg of a crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl) propionate (DTSSP; After stirring for 2 hours at 7 ° C, the mixture was ultrafiltered with an XM300 membrane and a CBS buffer (pH 8.5) to obtain 1 ml of liposome in which DTSSP was bound to HSA on the ribosome.
  • DTSSP crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl) propionate
  • Oligomannose 4 b Rokutokusari (Calbiochem Co., USA) 50 ⁇ g was added to 0. 5 ml aqueous solution prepared by dissolving NH 4 HC0 3 of 0. 25 g, After stirring for 3 days at 37 ° C, 0. 45 ⁇ The solution was filtered through a filter of ⁇ to complete the amination reaction of the reducing end of the sugar chain to obtain 50 g of an oligomannose 4b hexasaccharide glycosylamine compound. Next, the cross-linking reagent 3,3-dithiobis (sulfosucc inimi dyl) propionate was added to 1 ml of the liposome solution obtained in Example 3 for 1 minute.
  • ribosome (abbreviation: Man4b), in which oligomannose 4b hexasaccharide and human serum albumin and ribosome shown in FIG. 2 mg, total protein 200 ⁇ g, average particle size 100 nm) were obtained.
  • Example 11 Binding of oligomannose 5 heptasaccharide to ribosome membrane-bound human serum albumin (HSA) ''
  • Oligomannose pentasaccharide (Calbiochem Co., USA) (50 ⁇ g) was added to a 0.5 ml aqueous solution of 0.25 g of NH 4 HC0, and the mixture was stirred at 37 ° C. for 3 days. Then, the amination reaction of the reducing end of the sugar chain was completed to obtain 50 g of a glycosylamine compound having 5 oligosaccharides of oligomannose.
  • DTSSP crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl) propionate
  • DTSSP crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl) propionate
  • Oligomannose 6 Bruno, sugar (Calbiochem Co., USA) 50 ⁇ g was added to NH 4 HC0 3 of 0. 25 g to 0. 5 ml aqueous solution Dissolve 37. After stirring at C for 3 days, the mixture was filtered through a 0.45 ⁇ m filter to complete the amination reaction of the reducing end of the sugar chain, thereby obtaining 50 ⁇ g of an oligomannose 6-octasaccharide glycosylamine compound. Next, a crosslinking reagent 3, 3′-dithiobis (sulfosuccinimidyl) propionate (DTSSP;
  • Oligomannose 7 Kyutokusari (Calbiochem Co., USA) 50 g was added to 0. 5 ml aqueous solution of N HC0 3 was Dissolve the 0. 25 g, After stirring for 3 days at 37 ° C, filter 0. 45 m Then, the amination reaction of the reducing end of the sugar chain was completed to obtain 50 ⁇ g of a glycosylamine compound of oligomannose 7-nine sugar chain. Next, lmg of the crosslinking reagent 3,3'-dithiobis (sulfosuccinimi dyl) propionate (DTSSP; Pierce Co., USA) was added to a part of the liposome solution obtained in Example 3 at 25 ° C.
  • DTSSP crosslinking reagent 3,3'-dithiobis (sulfosuccinimi dyl) propionate
  • the mixture was stirred at 7 ° C and ultrafiltered with an XM300 membrane and a CBS buffer ( ⁇ 8.5) to obtain 1 ml of liposome with DTSSP bound to HSA on the ribosome.
  • the glycosylamine compound 50 of the oligomannose 79-sugar chain was added to the ribosome solution, and the mixture was stirred at 25 ° C for 2 hours, and then stirred at 7 ° C for 20 hours, and then the XM300 membrane and PBS buffer were added.
  • the solution was ultrafiltered with a solution (pH 7.2) to bind oligomannose 7-nine sugar chains to DTSSP on human serum albumin bound to the liposomal membrane.
  • the ribosome (abbreviation: Man7) in which the oligomannose 7-sugar chain, human serum albumin and ribosome shown in FIG. lOOnm).
  • Oligomannose 8 Jutokusari (Calb iochem Co., USA) 50 ⁇ g was added to 0. 5IO1 aqueous solution Dissolve the NH 4 HC0 3 of 0. 25 g, After stirring for 3 days at 37 ° C, in the filter After filtration, the amination reaction of the reducing end of the sugar chain was completed to obtain 50 g of a glycosylamine compound having oligomannose octasaccharide.
  • DTSSP 3,3′-dithiobis (sulfosuccinimi dyl) propionate
  • Oligomannose 9 H sugar (Calbiochem Co., USA) 50 ⁇ g was added to 0. 5 ml aqueous solution prepared by dissolving NH 4 HC0 3 of 0. 25 g, After stirring for 3 days at 37 ° C, 0. 45 ⁇ m The solution was filtered through the filter described in (1) to complete the amination reaction of the reducing end of the sugar chain to obtain 50 ⁇ g of a glycosylamine compound of an oligomannose 9H sugar chain.
  • oligomannose ⁇ -sugar chains were bound to DTSSP on human serum albumin bound to the liposome membrane by ultrafiltration with XM300 membrane and PBS buffer (pH 7.2).
  • 2 ml of ribosome (abbreviation: Man 9) in which oligomannose 9H sugar chain, human serum albumin and ribosome shown in 2 are linked (total fat mass 2 mg, total protein amount 200 g , average particle size lOOnm) was.
  • a crosslinking reagent 3 3'-dithiobis (sulfosuccinimidyl) propionate was added to 1 ml of the ribosome solution obtained in Example 3
  • TRIS ribosome
  • Example 17 Hydrophilization treatment on ribosome membrane surface-bound human serum albumin (HSA)
  • HSA protein surface on the liposome was hydrated by the procedure. 1 Separately, add 13 mg of tris (hydroxymethyl) aminomethane to 2 ml of two kinds of sugar chain-bound ribosomes, stir at 25 ° C for 2 hours, and then stir at 7 ° C.
  • the unreacted substances were removed by ultrafiltration with a buffer solution (pH 7.2), and the final product, the hydrated 12 sugar chain-binding ribosome complex (abbreviation: 'A2, A3 , .A4, A6, A36, Man3 Man4, Man5, Man6, Man7, Man8, Man9) were obtained in 2 ml each.
  • 'A2, A3 , .A4, A6, A36, Man3 Man4, Man5, Man6, Man7, Man8, Man9 were obtained in 2 ml each.
  • lectin-immobilized microplate lectin (Con A; R & D Systems Co., USA) was fixed on a 96-well microplate.
  • This lectin-immobilized plate, with Fuechuin 0. lg was bi Ochin of a comparative ligand, various different carbohydrate binding liposome complexes density (as protein amount, 0. 01 / ig, 0. 04 ⁇ ⁇ , 0.0.33 / g, 1 ⁇ g) and incubated at 4 ° C for 2 hours. After washing three times with PBS (P H 7.
  • Example 1 9 1251 labeling of various sugar chain-binding ribosomes by the chloramine T method
  • Chloramine T (Wako Pure Chemical Co., Japan) solution and sodium disulfite solution were prepared and used at 3 mg / ml and 5 mg / ml, respectively. Placed in a 1 two sugar chain binding ribosome and tri s (hydroxymethyl) aminomethane bonded Rihoso Ichimu and 50 Eppe down tube to one ⁇ Ri ⁇ prepared in 1 6 Example 4, followed by 125 preparative Nal (NEN Life Science Product, Inc. USA) was added to 15 ⁇ l, and chloramine T solution was added at 10 t 1 to react. Every 5 minutes, 10 ml of chloramine T solution was added, and this operation was repeated twice. After 15 minutes, sodium disulfite ( ⁇ ) was added as a reducing agent to stop the reaction.
  • Ehrlich ascites tumor (EAT) cells (approximately 2 x 10 7 cells) were transplanted subcutaneously into male ddY mice (7 weeks old), and the cancer tissue grew to 0.3 to 0.6 g (after 6 to 8 days) This was used in this experiment.
  • the tumor-bearing mouse was injected into the tail vein at a rate of 3 g / mouse using 0.2 ml of the liposome complex conjugated with 12 kinds of sugar chains and tris (hydroxymethyl) aminomethane as labeled in Example 19.
  • the tissue blood, liver, spleen, lung, brain, cancer tissue, inflamed tissue around cancer, lymph nodes
  • a gamma counter Aloka ARC 300
  • the amount of radioactivity distributed to each tissue was expressed as the ratio of radioactivity per lg of each tissue to the total radioactivity administered (% dose-amount / g tissue). The results are shown in FIGS.
  • Example 3 1 ml of the liposome solution obtained in Example 3 was added to 1 ml of the crosslinking reagent 3,3'-dithiobis (sulf osuccinimidylj propionate (DTSSP; Pierce Co., USAJlmg) for 2 hours at 25 ° C, followed by 7 ° C The mixture was ultrafiltered with an XM300 membrane and a CBS buffer (pH 8.5) to obtain 1 ml of liposomes in which DTSSP was bound to HSA on the liposomes.
  • DTSSP crosslinking reagent 3,3'-dithiobis (sulf osuccinimidylj propionate
  • -dithiobis sulfosuccinimidyl repropionate (DTSSP; Pierce Co., USA) lmg and heat at 25 ° C for 2 hours, then 7 ° C for 1 hour, limit with XM300 membrane and CBS buffer (pH 8.5) After filtration, 1 ml of ribosome in which DTSSP was bound to HSA on the ribosome was obtained, and the ribosome solution was added to the glycosylamido of the 3′-sialyllactosamine sugar chain described above.
  • DTSSP sulfosuccinimidyl repropionate
  • the mixture was stirred for 2 hours at 7 ° C., then stirred at 7 ° C., and subjected to ultrafiltration with an XM300 membrane and a CBS buffer (pH 8.5) to obtain 1 ml of ribosome in which DTSSP was bound to HSA on the ribosome.
  • Example 25 Hydrophobizing treatment on human serum albumin (HSA) bound to ribosome membrane surface
  • Example 2 Separately from the liposomes to which each of the two types of sugar chains had been bound prepared by means of 1 to 24, respectively. Then, the HSA protein surface on the ribosome was subjected to a hydrophilic treatment by the following procedure. 1 Separately into 2 ml of two types of sugar chain-linked ribosomes, Add 13mg of tris (hydroxymethyl) aminomethane, 2 hours at 25 ° C, then 7. After overnight stirring at C, the unreacted substances were removed by ultrafiltration with XM300 membrane and PBS buffer (pH 7.2).
  • Ribosome complex (abbreviation: 3SL-2, 3SL-2, 3SL-3, 6SL-1, 6SL-2, 6SL-3, 3SLN-1, 3, SLN-2, 3SLN-3, 6SLN-1, 6SLN-2 , 6SLN-3) 2 ml each (total lipid 2 mg, total protein 200 ⁇ g, average particle size 100 nm).
  • Example 2 The in vitro lectin-binding activity of each of the 12 types of sugar chain-binding ribosome complexes prepared by means of! To 24 and Example 16 was determined by a conventional method (Yam1 ⁇ 2aki, N. (1999) Drug According to the Delivery System, 14, 498-505), it was measured in an inhibition experiment using a lectin-immobilized microphone p-plate. That is, lectin (E-selectin; R & D Systems Co., USA) was fixed on a 96-well microplate. The lectin fixed 3 ⁇ 4 (the spoon plates, with fucosylated Fuechuin 0. 1 g was Biochin of a comparative ligand, as different various carbohydrate binding ribosome complex (protein amount of concentration, 0.
  • Chloramine T. (Wako Pure Chemical Co., Japan) Dissolution solution and sodium nitrite: The sodium ⁇ acid solution was prepared and used at 3 mg / ml and 5 mg / ml, respectively.
  • Example 21 One to three types of sugar chain-binding ribosomes and tris (hydroxymethyl) amidan ethane-binding liposomes prepared in accordance with Examples 1 to 24 and Example 16 were separately placed in an Eppendorf tube, and then successively placed in an Eppendorf tube. The reaction was performed by adding 15 ⁇ l of 1251-NaI (solid Life Science Product, Inc. USA) and 10 ⁇ l of a chloramine T solution. Every 5 minutes, Kulamin (solution) ( ⁇ ⁇ ) was added, and this operation was repeated twice.
  • 1251-NaI solid Life Science Product, Inc. USA
  • Example 28 Measurement of the amount of various sugar chain-linked ribosome complexes transferred from intestinal tract to blood in mice
  • Ribosomes were prepared using cholate dialysis. That is, dipalmitoyl phosphatidinorecholine, cholesterol monophosphate, disetinolephosphophate ', gangli. Sid and dipalmitoyl phosphatidylethanolamine in a molar ratio of 35: 40: 5: 15: 5, respectively. The mixture was mixed so that the total lipid amount was 45.6 mg, and 46.9 mg of sodium cholate was added, and dissolved in 3 ml of a chloroform / methanol solution. The solution was evaporated, and the precipitate was dried in vacuum to obtain a lipid membrane.
  • the obtained lipid membrane was suspended in 10 ml of a TAPS buffered physiological saline (pH 8.4) and sonicated to obtain 10 ml of a transparent micelle suspension.
  • the doxorubicin, an anticancer drug completely dissolved in TAPS buffer (pH 8.4) to a concentration of 3 mg / l ml, is slowly added dropwise to the micelle suspension with stirring, and the mixture is uniformly mixed.
  • the solution was subjected to ultrafiltration using PM10 membrane (Amicon Co., USA) and TAPS buffered saline ( ⁇ 8.4) to prepare 10 ml of a uniform suspension of ribosome particles containing doxorubicin, an anticancer drug.
  • the particle size and zeta potential of the anticancer drug doxorubicin-encapsulated ribosome particles in the obtained saline suspension (37 ° C) were measured using the zeta potential “particle size” molecular weight analyzer (Model Nano ZS, Malvern Instruments Ltd., UK) As a result, the particle diameter was 50 to 350 nm and the zeta potential was 130 to 110 mV.
  • Example 30 Hydrophilization treatment on anti-cancer drug doxorubicin-encapsulated ribosome lipid membrane 10 ml of the anticancer drug doxorubicin-encapsulated liposome solution prepared in Example 29 was mixed with XM300 membrane '(Amicon Co., USA) and CBS buffer (pH 8.5). ) was subjected to ultrafiltration, and the ⁇ of the solution was increased to 8.5 ⁇ .
  • the liposome membrane encapsulating the anticancer drug doxorubicin was hydrophilized on the lipid dipalmitoylphosphatidylethanolanolamine by the hydroxyl group power of tris (hydroxymethyl) aminomethane.
  • HSA human serum albumin
  • Alpha 1, 6 mannobiose disaccharide (Calbiochem Co., USA) 50 g was added to 0. 5 ml aqueous solution prepared by dissolving the N HC0 3 of 0. 25 g, After stirring for 3 days at 37 ° C, of 0. 45 m The resulting solution was filtered through a filter to complete the amination reaction of the reducing end of the sugar chain to obtain 50 g of a dalicosylamine compound having an alpha 1,6 mannobiose disaccharide chain.
  • Example 31 To 1 ml of the obtained anticancer drug doxorubicin-encapsulated ribosome solution, add 1 mg of the crosslinking reagent 3,3'-dithiobis ⁇ sulfosuccinimi dyl) propionate (DTSSP; Pierce Co., USA) at 25 ° C for 2 hours, followed by 7 ° C The mixture was stirred overnight at C and ultrafiltered with an XM300 membrane and CBS buffer (pH 8.5) to obtain 1 ml of ribosome in which DTSSP was bound to HSA on the liposome.
  • DTSSP 3,3'-dithiobis ⁇ sulfosuccinimi dyl) propionate
  • glycosyl Amin compound 50 mu ⁇ of the alpha 1,6 Man'nobiosuni sugar chains was stirred for 2 hours at 25 ° C, followed by 7 ° C De ⁇ , using an XM 300 membrane and a PBS Ultrafiltration with a buffer solution (pH 7.2) was performed to bind alpha 1,6-mannobiose disaccharide to DTSSP on human serum albumin bound to the liposomal membrane.
  • 13 mg of tris (hydroxymethyl) aminomethane (Wako Co., Japan; 13 mg) was added to the liposome solution, and the mixture was stirred at 25 ° C for 2 hours, and then stirred at 7 ° C for 10 minutes.
  • the particle size and zeta potential of the anticancer drug doxorubicin-encapsulated liposome particles in the obtained physiological saline suspension (37 ° C) were measured using a zeta potential, particle size, and molecular weight measurement device (Model Nano ZS, Malvern Instruments Ltd., UK). As a result of measurement, the particle diameter was 50 to 350 nm, and the zeta potential was -30 to 110 mV.
  • Example 31 1 ml of the anticancer drug doxorubicin-encapsulated liposomal solution obtained in Example 31 was added to 1 ml of the crosslinking reagent 3, 3, and Add lmg of -dithiobis (sulfosuccinimidyl) propionate (DTSSP; Pierce Co., USA) and stir at 25 ° C for 2 hours, then at 7 ° C, and mix with XM300 membrane and CBS buffer (pH 8.5). Ultrafiltration yielded 1 ml of liposomes with DTSSP bound to HSA on the liposomes.
  • DTSSP -dithiobis (sulfosuccinimidyl) propionate
  • ribosomes 2 ml of ribosomes (abbreviation: DX-3FL), an anticancer drug that has been treated with hydrophilicity of linker protein (HSA) in which 3-fucosyllactose trisaccharide, human serum albumin, and liposome are bound.
  • HSA linker protein
  • 2 mg of total lipid and 200 g of total protein were obtained in the obtained physiological saline suspension (37 ° C).
  • the particle size and zeta potential of the anticancer drug doxorubicin-encapsulated liposome particles were measured using a zeta potential ⁇ particle size and molecular weight measurement device (Model Nano ZS, Malvern Instruments Ltd., UK). The particle size was 50 to 350 nm and the zeta potential was It was one thirty to one ten mV.
  • Example 3 Hydrophilization treatment of linker protein (HSA) by binding of tris (hydroxymethyl) aminomethane to human serum albumin (HSA) bound to ribosome membrane containing anticancer drug doxorubicin
  • a cross-linking reagent was added to 1 ml of the ribosome solution containing the anticancer drug doxorubicin obtained in Example 31.
  • doxorubicin-encapsulated ribosome (abbreviated as abbreviated name) was used as a comparative sample, as shown in Fig. 32, in which a linker protein (HSA) in which tris (hydroxymethyl) aminomethane, human serum albumin, and liposomes were bound was made hydrophilic. : DX-TRIS) 2 ml (total lipid 2 mg, total protein 200 / g) was obtained.
  • the particle size and zeta potential of the anticancer drug doxorubicin-encapsulated ribosome particles in the obtained physiological saline suspension (37 ° C) were measured using a zeta potential, particle size, and molecular weight measurement device (Model Nano ZS, Malvern Instruments Ltd., UK). As a result of the measurement, the particle diameter was 50 to 350 nm, and the zeta potential was 13010 mV.
  • Ehrlich ascites tumor (EAT) cells (approximately 2 x 10 7 cells) were transplanted subcutaneously into the right thigh of male ddY mice (7 weeks old), and the cancer tissue grew to 50 to 100 cubic millimeters (6 Approximately 40 days later, about 40 mice were used in this experiment, and the 10 cancer-bearing mice were divided into four groups, each of which was divided into four groups, and the sugar chain-linked anticancer agent prepared in Examples 32 and 34.
  • Doxorubicin-encapsulated ribosome solution or sugar-free anticancer drug Doxorubicin-encapsulated ribosome solution or saline or free doxorubicin solution 0.2 ml, 4 times every 3 to 4 days (7, 11, 14 days after cancer cell transplantation)
  • doxorubicin-encapsulated ribosome quantification of encapsulated drug and storage stability-The ribosome was prepared using cholate dialysis. That is, dipalmitoylphosphatidinolecholine, cholesterol monophosphate, dicetinolephosphate, gangli-side, and dipalmitoylphosphatidylethanolamine in a molar ratio, respectively.
  • the mixture was mixed at a ratio of 35: 40: 5: 15: 5 to a total lipid amount of 45.6 mg, added with 46.9 mg of sodium cholate, and dissolved in 3 ml of a formaldehyde methanol solution. The solution was evaporated, and the precipitate was dried in vacuum to obtain a lipid membrane.
  • the obtained lipid membrane was suspended in 3 ml of a TAPS buffer (pH 8.4) and sonicated to obtain 3 ml of a transparent micelle suspension. To this micelle suspension, add PBS buffer (pH 7.2) to make 10 ml, and then stir the doxorubicin completely dissolved in TAPS buffer (pH 8.4) to 3 mg / l ml while stirring.
  • the doxorubicin-containing micellar suspension was subjected to ultrafiltration using PM10 membrane (AmiconCo., USA) and TAPS buffer (pH 8.4), and the doxorubicin-encapsulated ribosome particles were uniformly mixed. A 10 ml suspension was prepared. The particle size and zeta potential of the anticancer drug doxorubicin-encapsulated ribosome particles in the obtained physiological saline suspension (37 ° C) were measured using a zeta potential, particle size, and molecular weight measurement device (Model Nano ZS,
  • the average particle size was 50 to 350 nm, and the average zeta potential was 130 to 110 mV. Measure the amount of drug encapsulated in this liposome
  • doxorubicin When measured at 485 nm, it was found that doxorubicin was encapsulated at a concentration of about 110 ⁇ ug / ml. This doxorubicin-encapsulated ribosome was stable without precipitation or aggregation even after storage in a refrigerator for one year.
  • anti-cancer agent doxorubicin encapsulated ribosome solution 10ml the XM300 membrane prepared in (Ami con Co., USA) and were 8.5 to P H of the solution subjected to ultrafiltration using a CBS buffer solution (pH 8.5).
  • 10 ml of a crosslinking reagent bis (sulfosuccinimidyl) suberate (BS3; Pierce Co., USA) was added, and the mixture was stirred at 25 ° C for 2 hours. Thereafter, the mixture was further stirred at 7 ° C. for one minute to complete the chemical bonding reaction between the lipid dipalmitoylphosphatidylethanolamine on the ribosome membrane and BS3.
  • the ribosome solution was subjected to ultrafiltration using an XM300 membrane and a CBS buffer (pH 8.5).
  • a CBS buffer pH 8.5
  • 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of CBS buffer (pH 8.5) was calo-extracted into 10 ml of ribosome solution, stirred at 25 ° C for 2 hours, and stirred at 7 ° C overnight, followed by ribosome
  • the binding reaction between BS3-bound to lipid on the membrane and tris (hydroxymethyl) aminomethane was completed.
  • HSA human serum albumin
  • the particle size and zeta potential of the anticancer drug doxorubicin-encapsulated ribosome particles with a sialyl Lewis X-type tetrasaccharide in the obtained physiological saline suspension (37 ° C) were measured by using a zeta potential, a particle size, and a molecular weight measurement device (Model Nano ZS, As a result of measurement by Malvern Instruments Ltd., UK), the particle size was 50 to 350 nm, and the zeta potential was 130 mV.
  • Doxorubicin-encapsulated ribosome as a comparative sample (without sugar chain)
  • a cross-linking reagent 3,3′-dithiobis (sulfosuccinimidyl) propionate (DTSSP;) is added to a part (1 ml) of the anticancer drug doxorubicin-encapsulated ribosome solution obtained in (3).
  • the particle size and zeta potential of the anticancer drug doxorubicin-encapsulated ribosome particles (without sugar chains) in the obtained physiological saline suspension (37 ° C) were measured using a zeta potential, particle size, and molecular weight measurement device (Model Nano ZS). , Malvern Instruments Ltd., UK), the particle size was 50 to 350 nm, and the zeta potential was -30 to 110 mV.
  • the ribosome produced in this example was used in Example 38.
  • Example 3 8 Examination of leukemia treatment by targeting ribosome
  • KG-la cells were used, and the cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • MRC5 cells established from normal fibroblasts are used as normal human cells, and these cells contain 10% FBS.
  • the cells were cultured in MEM medium. Create a cell suspension of 105 cells / ml in these cells were cultured seeded 10 4 cells / well in 96-Ueru culture plates.
  • Free doxorubicin hydrochloride hereinafter free Dox
  • L- Dox ribosomized doxorubicin hydrochloride that has no sugar chain attached
  • ribosomized doxorubicin hydrochloride is sialyl Lewis X sugar Chain-bound (L-Dox-SLX) was administered to each well at an appropriate concentration. At this time, the concentration of FBS etc. Adjusted to equal conditions for each pell.
  • the surviving cells were quantified, the killing activities were calculated, and the 50% inhibitory drug concentration (50 ° /. Growth inhibitory ion concentration or less IC50) was calculated. I asked. For cell quantification, remove the medium containing the anticancer drug, replace with 100 ⁇ l RPMI 1640 medium or MEM medium containing 10% WST-8 and 10% FBS, and culture for 3 hours. It was measured (the absorbance at 550 nm was used as a reference value).
  • Killing activity (%) 100-(Attention 485 nm absorbance of the well-550 nm absorbance of the same well) I (Untreated 485 nm absorbance of the well-550 nm absorbance of the untreated well) X 100 (%) (Hereinafter referred to as KA).
  • Cytotoxicity was expressed as 50% inhibitory drug concentration (IC50) (uM).
  • MRC5 cells were used as normal human cells and KG-la cells were used as human leukemia cells From the results, the following was found.
  • Ribosomeation of doxorubicin hydrochloride reduced its toxicity to normal cells by about 62-fold with L-Dox and about 95-fold with L-Dox-SLX. This indicates that ribosome formation greatly reduced the toxicity of anticancer drugs.
  • the current dosing regimen of doxorubicin hydrochloride 0.4 to 0.6 mg / Kg (0.67 to 1 uM) per day for several days, the IC50 for normal cells A value of 500 ⁇ or more is considered very safe.
  • L-Dox-SLX provides high blood retention and low cytotoxicity to normal cells by ribosome formation, and is ideal for active accumulation on leukemia cells due to attached sugar chains. It seemed to be directional DDS.
  • the KA of IFN itself to KG-la cells was 9.4% ⁇ 7.34 with 100 U / ml of IFN added to the medium.
  • the KA of L-Dox and L-Dox-SLX was compared with 100 U / ml of IFN added to the medium (FIG. 43).
  • the doxorubicin hydrochloride concentration in L-Dox and L-Dox-SLX was 17.8 M.
  • L-Dox KA was only enhanced to 10% ⁇ 10, 5 without IFN and to 19.2% ⁇ 7.99 with IF added.
  • the KA of IFN itself is 9.4%
  • the enhancement of L-Dox by the addition of IFN was simply additive.
  • the KA of L-Dox-SLX is 6.3 ° / without IFN. ⁇ 11.6, 44.1 ° / with IFN added. Increased to ⁇ 3.76. This increased "" strong effect was very large and synergistic.
  • Neutralizing antibodies against L-selectin were added to the medium at a concentration of 0.3 / ig / ml, and KA of the antibodies alone was measured.
  • the antibody had a KA of 16.3% ⁇ 25.8 at a concentration of 0.3 i / ml.
  • the KA of L-Dox-SLX (doxorubicin hydrochloride concentration: 17.8 ⁇ M) with IFN added was 47.2% ⁇ 3.71, but when a neutralizing antibody was added to this state, ⁇ was 23. It was suppressed to 2% ⁇ 12.7 (Fig. 44). Considering the ⁇ of the neutralizing antibody itself, it was thought that ⁇ of L- Dox-SLX was almost completely suppressed when IFN was added.
  • Liposomes were prepared using the cholate dialysis method. That is, dipalmitoylphosphatidinorecholine, cholesterol monophosphate, dicetinolephosphate, gangliside (GM1: 13 ° / D, GDla: 38 %, GDla: 9 ° /., GDlb: 9 ° /., GTlb: 1 ⁇ %) in a molar ratio of 35: 45: 5: 15, respectively, to a total lipid amount of 45.6 m, add 46.9 mg of sodium cholate, and 3 ml of chloroform / methanol solution Dissolved in The solution was evaporated and the precipitate was dried in vacuo to give a lipid membrane.
  • the obtained lipid membrane was suspended in 3 ml of TAPS buffer ( ⁇ 8.4) and sonicated to obtain 3 ml of a transparent micelle suspension.
  • TAPS buffer ⁇ 8.4
  • PBS buffer pH 7.2
  • doxorubicin completely dissolved in TAPS buffer ( ⁇ 8.4) to 3 mg / l ml while stirring.
  • this micelle suspension containing doxorubicin was subjected to ultrafiltration using PM10 membrane (AmiconCo., USA) and TAPS buffer (pH 8.4) to obtain a uniform glycolipid-type sugar.
  • a 10 ml suspension of doxorubicin-encapsulated liposome particles having a chain was prepared.
  • the particle diameter and zeta potential of the anticancer drug doxorubicin-encapsulated ribosome particles having glycolipid-type sugar chains in the obtained physiological saline suspension (37 ° C) were measured.
  • the particle size was 50 to 350 nm, and the zeta potential was 130 to 10 mV.
  • the amount of the drug encapsulated in the ribosome was measured at an absorbance of 485 nm, it was found that doxorubicin was encapsulated at a concentration of about 71 / _ig / ml. This doxorubicin-encapsulated ribosome was stable without precipitation or aggregation even after storage in a refrigerator for one year.
  • Ehrlich ascites tumor (EAT) cells (approximately 2 x 10 7 cells) were implanted subcutaneously into the right thigh of male ddY mice (7 weeks old), and the cancer tissue became 50 to 100 cubic millimeters. Approximately 50 animals after 8 days) were used in this experiment. The 25 cancer-bearing mice were divided into two groups and transplanted with cancer cells in each group. . Thereafter, the concentration of doxorubicin in the blood or tumor tissue of 5 animals in each group was measured by fluorescence method (470 nm) over time. In addition, the area under the time curve of drug blood concentration (AUC) and the distribution of doxorubicin in tumor tissues and cells were observed with a fluorescence microscope.
  • AUC drug blood concentration
  • the doxorubicin in the blood when the doxorubicin encapsulated in the liposome of the present invention was used compared to the conventional free doxorubicin. It was found that the retention was about 100 times higher, and the doxorubicin accumulation effect on tumor tissues and cancer cells was about tens of times higher.
  • Ehrlich ascites tumor (EAT) cells (approximately 2 x 10 7 cells) were implanted subcutaneously into the right thigh of male ddY mice (7 weeks old), and cancer tissues grew to 50 to 100 cubic millimeters (6 to 8 cubic millimeters). About 30 days later) were used in this experiment. After dividing the 10 tumor-bearing mice into 3 groups and transplanting the cancer cells into each group, 0.2 ml of a doxorubicin-encapsulated liposome solution having a glycolipid-type sugar chain or free doxorubicin solution or physiological saline was added. Intravenous injection was performed 6 times every 4 days.
  • Ribosomes were prepared using cholate dialysis. That is, dipalmitoylphosphatidinorecholine, cholesterol monophosphate, dicetinolephosphate, gandarioside, and dipalmitoylphosphatidylethanolamine in a molar ratio of 35: 40: 5: 15: 5, respectively.
  • the mixture was mixed to an amount of 45.6 mg, added with 46.9 mg of sodium cholate, and dissolved in 3 ml of a formaldehyde methanol solution. The solution was evaporated, and the precipitate was dried in vacuum to obtain a lipid membrane.
  • the obtained lipid membrane was suspended in 3 ml of a TAPS buffer (pH 8.4) and sonicated to obtain 3 ml of a clear micelle suspension.
  • a TAPS buffer pH 8.4
  • PBS buffer ⁇ 7.2
  • TAPS buffer pH 8.4
  • the micelle suspension containing prednisolone phosphate is subjected to ultrafiltration using PM10 membrane (AmiconCo., USA) and TAPS buffer (pH 8.4) to homogenize. 10 ml of fresh ribosome was prepared.
  • Obtained saline suspension The particle size and zeta potential of the liposome particles in the suspension (37 ° C) were measured with a zeta potential, particle size and molecular weight measurement device (Model Nano ZS, Malvern Instruments Ltd., UK). 350350 nm, the zeta potential was ⁇ 30 to ⁇ 10 mV.
  • the ribosome solution was subjected to ultrafiltration using an XM300 membrane and a CBS buffer (pH 8.5).
  • 50 mg of cellobiose dissolved in 1 ml of CBS buffer (pH 8.5) was added to 10 ml of the liposome solution, and the mixture was stirred at 25 ° C for 2 hours, and then stirred at 7 ° C overnight to remove lipids on the ribosome membrane.
  • the chemical bond reaction between the bound BS3 and cellobiose was completed.
  • the hydroxyl group of cellobiose was coordinated on the lipid dipalmitoylphosphatidylethanolamine of the ribosome membrane to make it hydrated and hydrophilic.
  • HSA human serum albumin
  • HSA human serum albumin
  • HSA human blood albumin
  • Total lipid amount 2 mg, total protein amount 200 g was obtained.
  • the particle size and zeta potential of the ribosome particles in the obtained physiological saline suspension (37 ° C) were measured using the zeta potential
  • the particle diameter was 50 to 350 nm, and the zeta potential was 130 to 10 mV.
  • Ultrafiltration with an XM300 membrane and CBS buffer (pH 8.5) yielded 1 ml of liposomes treated with hydrophilicity of the linker protein (HSA) with DTSSP bound to HSA on the liposomes.
  • HSA linker protein
  • ribosome As a result, 2 ml of ribosome (total lipid amount: 2 mg, total protein amount: 200 ⁇ ) was used as one of the samples for hydrophilizing the linker protein (HSA) in which cellobiose, human serum albumin, and liposome were bound. g) was obtained.
  • the particle size and zeta potential of the ribosome particles in the obtained physiological saline suspension (37 ° C) were measured using a zeta potential, particle size, and molecular weight measurement device (Model Nano ZS, As a result of measurement by Malvern Instruments Ltd., UK), the particle size was 50 to 350 nm, and the zeta potential was 30 10 mV.
  • Ribosomes were prepared using cholate dialysis. That is, dipalmitoylphosphatidylcholine, cholesterol monophosphate, dicetinolephosphate, and gandarioside (containing 100% GTlb as glycolipid bran chain) in a molar ratio of 35: 45: 5: 15, respectively. The mixture was mixed so that the total lipid amount was 45.6 mg, 46.9 mg of sodium cholate was added, and the mixture was dissolved in 3 ml of a methanol solution of formaldehyde Z. The solution was evaporated and the precipitate was dried in vacuo to give a lipid membrane.
  • cholate dialysis That is, dipalmitoylphosphatidylcholine, cholesterol monophosphate, dicetinolephosphate, and gandarioside (containing 100% GTlb as glycolipid bran chain) in a molar ratio of 35: 45: 5: 15, respectively.
  • the mixture was mixed so that the total lipid amount was 45.6 mg, 46.9 mg
  • the obtained lipid membrane was suspended in 3 ml of a TAPS buffer (pH 8.4) and sonicated to obtain a clear micelle suspension (ml).
  • a TAPS buffer pH 8.4
  • sonicated 3 ml
  • PBS buffer pH 8.4
  • doxorubicin completely dissolved in TAPS buffer (pH 8.4)
  • TAPS buffer pH 8.4
  • the doxorubicin-containing suspension is subjected to ultrafiltration using PM10 membrane (AmiconCo., USA) and TAPS buffer (pH 8.4) to make it uniform and hydrophilic.
  • a 10 ml suspension of untreated ribosome particles was prepared.
  • the particle size and zeta potential of the liposome particles not subjected to hydrophilization treatment in the obtained suspension of physiological saline (37 ° C) were measured using the zeta potential, particle size and molecular weight measurement equipment (Model Nano ZS, Malvern Instruments Ltd , UK), the particle size was 50 to 350 nm, and the zeta potential was 130 to -10 mV.
  • Chloramine T (Wako Pure Chemical Co., Japan) solution and sodium disulfite solution were prepared and used at 3 mg / ml 'and 5 mg / ml, respectively.
  • Example 5 Separately put 50 1 each of the three ribosomes prepared in Examples 6 to 8 into an eppen tube, and then add 15 ⁇ l of 125 1-Nal (NEN Life Science Product, Inc. USA) and chloramine T solution. Was added and reacted. Chloramine solution was added every 5 minutes, and this operation was repeated twice. After 15 minutes, 100 ⁇ l of sodium disulfite was added as a reducing agent to stop the reaction. Next, the product was placed on a Sephadex G-50 (Phramacia Biotech.
  • Ehrlich ascites tumor (EAT) cells (approximately 2 x 10 7 cells) were transplanted subcutaneously into male ddY mice (7 weeks old), and the cancer tissue grew to 0.3 to 0.6 g (after 6 to 8 days) This was used in this experiment.
  • the injected dose in mice tail vein so that the ratio of 5 minutes after the blood - the cancer-bearing mice (9) by 30 / g / 125 1 labeled three ribosomes double coalescence 0. 2 ml as lipid amount
  • the samples were collected and their radioactivity was measured with a gamma counter (Aloka ARC 300).
  • the amount of radioactivity distributed to blood was expressed as the ratio of radioactivity per blood lffll to the total radioactivity administered (% dose 1 blood).
  • the two types of hydrophilization treatments improve the blood retention, and in particular, the improvement of the blood retention of ribosomes that have been hydrophilized with tris ('hydroxymethyl) aminomethane is evident. It was author.
  • Ribosomes were prepared using cholate dialysis. That is, dipalmitoyl phosphatidinorecholine, cholesterol, disetinolephosphophosphate, gangliosi, sphingomyelin, and dipalmitoyl phosphatidylethanolamine in a molar ratio of 35: 40: 5: 5 respectively.
  • the mixture was mixed at a ratio of 10: 5 to a total lipid amount of 45.6 mg, added with 46.9 mg of sodium cholate, and dissolved in 3 ml of a chloroform Z methanol solution. The solution was evaporated and the precipitate was dried in vacuo to give a lipid membrane.
  • the obtained lipid membrane was suspended in 3 ml of a PBS buffer (pH 7.2) and sonicated to obtain 3 ml of a transparent micelle suspension.
  • a PBS buffer pH 7.2
  • To this micelle suspension add PBS buffer (pH 7.2) to make 10 ml, and then add 0.3 ml of ethanol and 0.7 ml of PBS buffer (pH 7.2) to remove 6 mg of completely dissolved vitamin A. After slowly adding dropwise and stirring to mix uniformly, this micelle suspension containing vitamin A is subjected to ultrafiltration using PM10 membrane (Amicon Co., USA) and PBS buffer (pH 7.2) to homogenize. 10 ml of a new vitamin A-encapsulated ribosome (average particle size 100 nm) was prepared. This vitamin A-encapsulated ribosome was stable without precipitation or aggregation even after storage in a refrigerator] _ years.
  • Ribosomes were prepared using cholate dialysis. That is, dipalmi Phosphatidinorecolin, cholesterol monophosphate, dicetinolephosphate, gangli-side, sphingomyelin, and dipalmitoylphosphatidylethanolamine in a molar ratio of 35: 40: 5: 5: 10: 10: 5, respectively.
  • the mixture was mixed so that the total lipid amount was 45.6 mg, and 46.9 mg of sodium cholate was added, and 6 mg of vitamin E was also added, and the mixture was dissolved in 3 ml of a methanol solution of Cloz Form Z. The solution was evaporated and the precipitate was dried in vacuo to give a lipid membrane.
  • the obtained lipid membrane was suspended in 3 ml of a PBS buffer (pH 7.2) and sonicated to obtain 3 ml of a transparent micelle suspension.
  • a PBS buffer pH 7.2
  • PBS buffer ⁇ 7.2
  • PM10 membrane Amicon Co., USA
  • PBS buffer pH 7.2
  • Ultrafiltration was used to prepare 10 ml of uniform ribosomes (average particle size lOOnm) with vitamin A.
  • the vitamin E-encapsulated ribosome was stable without precipitation or aggregation even after storage in the refrigerator for one year.
  • Liposomes were prepared using the cholate dialysis method. That is, dipalmitoylphosphatidinolecholine, cholesterol monophosphate, dicetinolephosphate, gandarioside and dipalmitoylphosphatidylethanolamine in a molar ratio of 35: 40: 5: 15: 5, respectively.
  • the mixture was mixed to a lipid amount of 45.6 mg, added with sodium cholate (46.9 mg), and dissolved in chloroform Z methanol solution (3 ml). The solution was evaporated, and the precipitate was dried in vacuum to obtain a lipid membrane.
  • the obtained lipid membrane was suspended in 3 ml of TAPS buffer ( ⁇ ⁇ 8.4) and subjected to ultrasonic treatment to obtain 3 ml of a transparent micelle suspension.
  • TAPS buffer ⁇ ⁇ 8.4
  • PBS buffer pH 7.2
  • P H8.4 PBS buffer
  • the particle size and zeta potential of the prednisolone phosphate-encapsulated liposome particles in the obtained physiological saline suspension (37 ° C) were measured using the zeta potential, particle size, molecular weight measurement device (Model Nano As a result of measurement by ZS, Malvern Instruments Ltd., UK), the particle diameter was 50 to 350 nm, and the zeta potential was 130 to 10 mV. Measure the amount of drug encapsulated in this ribosome
  • prednisolone phosphate was encapsulated at a concentration of approximately 280 g / ml.
  • the prednisolone phosphate-encapsulated ribosome was stable without precipitation or aggregation even after storage in a refrigerator for one year.
  • the tris (hydroxymethyl) aminomethane was hydrated and hydrolyzed on the lipid dipalmitoyl phosphatidyl ethanoleamine on the lipid membrane of the anticancer drug doxorubicin-encapsulated ribosome.
  • HSA human serum albumin
  • HSA human serum albumin
  • the mixture was stirred at 7 ° C and ultrafiltered with an XM300 membrane and a CBS buffer (pH 8.5) to obtain 1 ml of ribosome in which DTSSP was bound to HSA on the ribosome.
  • 50 ⁇ g of the above-mentioned daricosylamine compound of the aryl Lewis X-type tetrasaccharide chain was added to the ribosomal solution, and the mixture was stirred at 25 ° C. for 2 hours, and then stirred at ⁇ 7 ° C. with the XM300 membrane.
  • ribosomes obtained by hydrolyzing linker protein (HSA), in which tris (hydroxymethyl) aminomethane, human blood albumin and liposomes were bound were obtained.
  • linker protein in which tris (hydroxymethyl) aminomethane, human blood albumin and liposomes were bound
  • the particle size and zeta potential of the prednisolone phosphate-encapsulated ribosome particles were measured using a zeta potential, particle size, and molecular weight measurement device (Model Nano ZS, Malvern Instruments Ltd., UK). At nm350 nm, the zeta potential was one 30 10 mV.
  • Hydrophilization treatment of linker protein (HSA) by binding of tris (hydroxymethyl) aminomethane onto human serum albumin (HSA) bound to prednisolone phosphate-encapsulated ribosome membrane
  • a cross-linking reagent 3 3'-dithiobis (sulfosuccinimidyl) was added to a part (1 ml) of the prednisolone phosphate-encapsulated ribosome solution obtained in (3).
  • prednisolone phosphate-encapsulated ribosomes saccharide
  • HSA diposomalized linker protein
  • the particle size and zeta potential of the prednisolone phosphate-encapsulated ribosome particles (without sugar chains) in the obtained physiological saline suspension (37 ° C) were measured using the zeta potential ⁇ particle size ⁇ molecular weight measurement device (Model Nano ZS, As a result of measurement by Malvern Instruments Ltd., UK), the particle diameter was 50 to 350 nm, and the zeta potential was -30 mV.
  • the ribosome prepared in this example was used in the examination of Examples 15 and 16 below.
  • a type II collagen-induced arthritis (CIA) model was created as an RA mouse model.
  • the mice and reagents used are as follows.
  • Inoculation antigen bovine collagen type II
  • Adjuvant complete Freund's adjuvant containing killed tuberculosis (H37Ra, 2 mg / ml) (CFA), incomplete Freund's adjuvant (IFA) containing no killed tuberculosis (IFA)
  • Collagen aqueous solution and CFA are mixed at a volume ratio of 2: 1. It was injected subcutaneously (Day 0). 21 days after treatment (Day 21) An aqueous solution of collagen and IFA were mixed at a volume ratio of 2: 1 to prepare an emulsion containing 100 / L of collagen and 200, and injected again subcutaneously at the bottom of the tail of the mouse.
  • mice The limbs of the mice were observed at a frequency of 3 times / week after the treatment, and the inflammation was quantified by pointing to the following criteria.
  • normal 0 points
  • mild inflammation or redness 1 point
  • severe redness suppression of swelling or use
  • 2 points 2 points
  • deformation of palms, soles and joints 3 points (minimum 0 points, The maximum is 12 points.)
  • IA Inflammatory Activity
  • mice with arthritis were treated by injection of a therapeutic agent via the tail vein. Intravenous injection was twice a week between Day 28 and Day 46.
  • mice with inflammatory findings were selected and divided into four groups so that the average inflammatory point in each group was the same.
  • Each group is 1) Control: no treatment group, 2) free
  • Pred prednisolone phosphate using an aqueous solution dissolved in saline, the amount of phosphoric acid prednisolone is once 100 ⁇ , week 200 mu g intravenously injected group, 3)
  • L-Pred follicle prednisolone phosphate ribosome Pre-built, use no sugar chain, prednisolone phosphate once a day ⁇ ⁇ ⁇ 20 g / wk group, 4)
  • L-Pred-SLX Phosphate Embedded prednisolone liposome, using those obtained by binding Shiariruruisu X sugar chain, the amount of prednisolone phosphate once 10 mu ⁇ , week 20 / g intravenously injected group was 4 groups.
  • mice in these four groups were Control: 6, free Pred: 7, L-Pred: 8, and L-Pred-SLX: 8. -The following results were obtained.
  • Control the IA increased over time, surpassing 60% as of Day 46.
  • free Pred after the IA reached about 50% on Day 37, it was in an equilibrium state.
  • L-Pred IA started to increase from Day 32 and was about 40% in Day 46. No significant increase in IA was observed for L_Pred_SLX, at 20 ° / throughout the course.
  • the results were as follows (Fig. 53). -From the results, the following was found.
  • the amount of prednisolone phosphate injected intravenously at 100 ⁇ g / injection was not enough to control inflammation, and no clear difference was observed in M compared to the control group.
  • the amount of intravenously injected prednisolone was 10 g / injection, one tenth of that in the free Pred group, but the IA was equal to or less than that in the free Pred group. This suggests that pred-zolone phosphate ribosome formation improved the drug's retention in blood, which may have improved drug efficacy.
  • L-Pred-SLX prednisolone phosphate amount in group was tenth of 10 ⁇ g / injection similarly free Pred group, IA inflammatory processes not at all increase was significantly suppressed. This was considered to be an effect brought about by the difference between L-Pred and L-Pred-SLX, and the presence or absence of sialyl Lewis X sugar chain. In other words, it was suggested that ribosomes could be effectively accumulated in inflammatory sites by sialyl Lewis X sugar chains.
  • This DDS proved to be able to distribute therapeutic agents to inflammatory sites very effectively. This suggests that by concentrating the therapeutic agent on the lesion in the treatment of inflammatory diseases, it is possible to suppress the side effects of the therapeutic agent or increase the efficacy of the therapeutic agent.
  • mice with arthritis were treated orally with a therapeutic agent. Dosing was performed three times a week from Day 28 to Day 39. On Day 28, mice with inflammation findings were selected and divided into four groups so that the average of the inflammation points in each group was almost the same. Each group consisted of 1) Control: no treatment group, 2) free Pred: prednisolone phosphate was used as an aqueous solution in physiological saline, and the dose of prednisolone phosphate was 100 / ig once a day, orally administered 300 ⁇ g / week.
  • L-Pred embedded-phosphate prednisolone ribosome, using what without a sugar, phosphorus sump Redonizoron quantities once 10 Ai g, week 30 mu ⁇ orally administered group
  • L-Pred-SLX Prednisolone phosphate embedded in liposomes, conjugated with sialyl Lewis X sugar chain, prednisolone phosphate dose of 10 / g at a time, 30 ⁇ weekly oral administration, There were 4 groups.
  • mice The number of mice in these four groups was Control: 2, free Pred: 3, L-Pred: 3, and L-Pred-SLX: 4.
  • the free Pred group died from gastric perforation due to the feeding needle, and was stopped during the experiment at Qay, 32. No significant difference in IA was observed between the control group, free Pred group (until Day 32), and L-Pred group. In the L-Pred-SLX group, the progression of inflammation could not be suppressed, but on Day 39, the IA was significantly lower than that of L-Pred (Fig. 54).
  • prednisolone phosphate could be transported to the inflamed site in the form of liposomes with sugar chains, given that the ribosome was absorbed in the intestinal tract and that the oral dose of prednisolone phosphate was 30 ⁇ g / week. I thought it was.
  • mice with inflammation findings were selected and divided into three groups so that the average of the inflammation points in each group was almost the same.
  • Each group was 1) untreated with Contro, 2) 250 ⁇ g: using an aqueous solution of prednisolone phosphate dissolved in physiological saline, and the amount of prednisolone phosphate was 250 ⁇ g once, 500 / g weekly intravenous injection 3)
  • 500 g a group in which an aqueous solution of prednisolone phosphate dissolved in physiological saline was used, and the amount of prednisolone phosphate was 500 g, and the group was injected intravenously 1,000 times a week.
  • mice in these three groups were Control: 2, 250 ⁇ g: 2, and 500 ⁇ g: 2.
  • ribosomes in which various glycans and a biologically-derived protein (linker) including human-derived proteins such as human serum albumin and ribosomes were produced, and various tissues in mice were produced. Analysis of the pharmacokinetics of ribosomes into cancer tissues, in particular, using Ehrlich solid tumor-bearing mice.
  • linker biologically-derived protein
  • the present invention very useful in the medical and pharmaceutical fields, it is possible to provide a ribosome capable of controlling the targeting.
  • a sugar chain-binding ribosome having higher target directivity could be obtained.
  • the sugar chain-modified ribosome of the present invention has an excellent intestinal absorption property, and is epoch-making in that it can be administered in a new dosage form that is not found in conventional liposome-based preparations by passing through the intestinal tract. It is something.
  • intestinal absorptive and various tissues blood, liver, spleen, lung, brain, small intestine, heart, thymus, kidney, Teng, muscle, large intestine, bone, bone marrow, eye, cancer tissue, inflamed tissue, lymph node
  • the pharmacokinetics of liposomes can be controlled by setting the amount of sugar chains bound to ribosomes and selecting the sugar chains, thereby efficiently and safely delivering drugs or genes to the intestinal tract and blood without any side effects. It is possible to transfer to living tissue via .This is particularly useful in the fields of medicine and pharmacy.
  • the sugar chain-modified hydrophilized ribosome and the non-sugar chain-modified hydrophilized liposome of the present invention are orally or parenterally administered to a subject carrying a cancer by encapsulating an anticancer agent, they are targeted to cancer tissues. Can accumulate and suppress cancer growth.
  • the targeting ribosome of the present invention can be used in tissues and organs expressing lectin capable of recognizing and binding to sugar chains bound to the ribosome surface in the body by encapsulating a compound having an appropriate pharmaceutical effect. A compound that reaches and is taken up by cells, where it has a medicinal effect, exerts its effect and can be used as a therapeutic or diagnostic agent.
  • an anticancer drug is encapsulated and can be used as a therapeutic drug for cancer.
  • the intestinal absorption-controlling ribosome of the present invention is easily absorbed from the intestinal tract, and a substance having a medicinal effect encapsulated in the body like the targeting ribosome can quickly exert its effect.

Abstract

L'invention concerne un liposome possédant une chaîne de sucre avec une activité de liaison spécifique à diverses lectines (protéines reconnaissant une chaîne de sucre) existant dans la surface cellulaire de divers tissus. Ainsi, une cellule ou un tissu in vivo peut être distingué et un médicament ou un gène peut être efficacement amené à cette cellule ou à ce tissu.
PCT/JP2004/011291 2003-08-01 2004-07-30 Liposome dirige sur une cible, enterique et a absorption commandee possedant une chaine de sucre, ainsi que remede contre le cancer contenant ce liposome et diagnostic mettant en oeuvre ce liposome WO2005011632A1 (fr)

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US10/566,566 US20070160657A1 (en) 2003-08-01 2004-07-30 Targeting and intestinal-absorption controlled liposome having sugar chain and therapeutic drug for cancer and diagnostic drug containing the liposome
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JPWO2007018272A1 (ja) * 2005-08-11 2009-02-19 独立行政法人産業技術総合研究所 糖鎖修飾リポソーム及び該リポソームを含有する薬剤送達組成物
JPWO2007018273A1 (ja) * 2005-08-11 2009-02-19 独立行政法人産業技術総合研究所 糖鎖修飾リポソームを含有する診断薬送達組成物
WO2007089043A1 (fr) * 2006-02-03 2007-08-09 Takeda Pharmaceutical Company Limited Preparation liposomique
WO2007091661A1 (fr) * 2006-02-08 2007-08-16 National Institute Of Advanced Industrial Science And Technology Liposome modifié par une chaîne de sucre convenant pour l'imagerie moléculaire et utilisation et production de celui-ci
JPWO2007091661A1 (ja) * 2006-02-08 2009-07-02 独立行政法人産業技術総合研究所 分子イメージングに適した糖鎖修飾リポソームならびにその利用および製造
JP2008179563A (ja) * 2007-01-24 2008-08-07 Cosmo Shokuhin Kk 有用リン脂質組成物を含む機能性素材及び機能性食品
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JPWO2009148169A1 (ja) * 2008-06-06 2011-11-04 片山化学工業株式会社 アンミン白金錯体を高濃度に内包するリポソームを用いた腫瘍治療技術
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US10918726B2 (en) 2014-11-28 2021-02-16 Quarrymen & Co. Inc. Carbosilane dendrimer and aggregatable carrier obtained using said dendrimer for drug delivery system
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WO2017204337A1 (fr) 2016-05-27 2017-11-30 国立大学法人埼玉大学 Capsule pour systèmes d'administration de médicament du type à administration spécifique au tissu cible utilisant un dendrimère de carbosilane
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EP1655038A4 (fr) 2010-06-02
EP1655038A1 (fr) 2006-05-10
US20070160657A1 (en) 2007-07-12

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